Anti-activin a antibodies and uses thereof

ABSTRACT

The present invention provides antibodies that bind to Activin A and methods of using the same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to Activin A with high affinity. The antibodies of the invention are useful for the treatment of diseases and disorders characterized by decreased muscle mass or strength, such as sarcopenia, cachexia, muscle injury, muscle wasting/atrophy, cancer, fibrosis, and weight loss. The antibodies of the invention are also useful in combination with Growth and Differentiation Factor 8 (GDF8) binding proteins for the treatment of diseases and disorders characterized by decreased muscle mass or strength. The antibodies of the invention are also useful for the prevention, treatment, or amelioration of disorders and diseases caused by, promoted by, exacerbated by, and/or aggravated by Activin A, such as renal fibrosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application No.16/926,481, filed Jul. 10, 2020, which is a continuation of U.S. Pat.Application No. 16/692,583, filed Nov. 22, 2019, abandoned, which is acontinuation of U.S. Pat. Application No. 15/637,867, filed Jun. 29,2017, now U.S. Pat. No. 10,526,403, issued Jan. 7, 2020, which is acontinuation of U.S. Pat. Application No. 14/447,444, filed Jul. 30,2014, now U.S. Pat. No.: 9,718,881, issued Aug. 1, 2017, which claimsthe benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos.61/859926, filed Jul. 30, 2013, 61/864036, filed Aug. 9, 2013,61/911834, filed Dec. 4, 2013, and 61/913885, filed Dec. 9, 2013, thedisclosures of each of which are herein incorporated by reference intheir entireties.

SEQUENCE LISTING

The instant application includes a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Nov. 17, 2022 isnamed Sequence-Listing-40848-0051 USC4 and is 280,409 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for Activin A, and methods of usethereof, including methods of using antibodies specific for Activin A inconjunction with a myostatin inhibitor.

BACKGROUND

Activins belong to the transforming growth factor-beta (TGF-β)superfamily and exert a broad range of biological effects on cellproliferation, differentiation, and apoptosis. Activins are homo- orheterodimers of InhibinβA, InhibinβB, InhibinβC and InhibinβE, differentcombinations of which create the various members of the activin proteingroup. For example, Activin A is a homodimer of InhibinβA and Activin Bis a homodimer of InhibinβB, whereas Activin AB is a heterodimer ofInhibinβA and InhibinβB and Activin AC is a heterodimer of InhibinβA andInhibinβC (Tsuchida, K. et al., Cell Commun Signal 7:15 (2009)).

Activin A binds to and activates receptor complexes on the surface ofcells known as Activin Type II receptors (Type IIA and Type IIB, alsoknown as ActRIIA and ActRIIB, respectively). The activation of thesereceptors leads to the phosphorylation of an Activin Type I receptor(e.g., Alk4 or 7), which in turn leads to the phosphorylation of SMAD 2and 3 proteins, the formation of SMAD complexes (with SMAD4), and thetranslocation of the SMAD complex to the cell nucleus, where SMAD2 andSMAD3 function to regulate transcription of various genes (Sozzani, S.and Musso, T., Blood 117(19):5013-5015 (2011)).

Numerous other ligands bind to and activate ActRIIB, including GDF8(myostatin), Activin B, Activin AB, Inhibin A, Inhibin B, GDF3, GDF11,Nodal, BMP2, BMP4, BMP7, BMP9, and BMP10. Blocking the interactions ofActRIIB with its ligands can lead to beneficial physiological effects.For example, GDF8 plays a central role in the development andmaintenance of skeletal muscle, acting as a negative regulator of musclemass (McPherron AC et al. (1997). Nature 387(6628):83-90).Administration of ActRIIB-Fc (i.e., the extracellular portion of theType IIB receptor, ActRIIB, stabilized by fusion to an IgG Fc domain)leads to significant increases in skeletal muscle mass and improvesmuscle weight and measurements of muscle strength in mice (Lee SJ, etal. (2005) Proc Natl Acad Sci U S A 102(50):18117-18122). The efficacyof ActRIIB-Fc is attenuated but not eliminated in Mstn (myostatin) nullmice, demonstrating that other ActRIIB ligand(s) in addition tomyostatin can function as negative regulators of muscle growth. Thus, aneed exists for additional inhibitors of ActRIIB signaling that canprovide clinical benefits.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that bind inhibin βA anddimers containing inhibin βA, e.g., Activin A, Activin AB, etc.. Theantibodies of the invention are useful, inter alia, for inhibitingActivin A-mediated signaling, producing beneficial clinical outcomesthrough the inhibition of Activin A-mediated signaling, e.g., fortreating diseases and disorders caused by or related to Activin Aactivity and/or signaling. The antibodies of the invention also haveutility for use in conjunction with inhibitors of other ligands of theActRIIA and ActRIIB receptors, such as GDF8 inhibitors.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., J Immunol 164:1925-1933 (2000)).

The present invention provides isolated antibodies, or antigen-bindingfragments thereof, that specifically bind Activin A with a bindingassociation equilibrium constant (K_(a)) of less than about 500 nM and adissociation equilibrium constant (K_(D)) of less than about 5 pM asmeasured in a surface plasmon resonance assay at 25° C. In someembodiments of the invention, the isolated antibodies, orantigen-binding fragments thereof, specifically bind Activin A with aK_(D) of less than about 4 pM as measured in a surface plasmon resonanceassay at 25° C. In some embodiments of the invention, the isolatedantibodies, or antigen-binding fragments thereof, specifically bindActivin A with a binding association equilibrium constant (K_(a)) ofless than about 500 nM.

The present invention provides isolated antibodies, or antigen-bindingfragments thereof, that specifically bind Activin A and block binding ofat least one Activin A receptor to Activin A. In some embodiments of theinvention, the isolated antibodies, or antigen-binding fragmentsthereof, block Activin A binding to an Activin A receptor with an IC₅₀value of less than about 80 pM as measured in an in vivo receptor/ligandbinding bioassay at 25° C. In some embodiments of the invention, theisolated antibodies, or antigen-binding fragments thereof, block ActivinA binding to an Activin A receptor with an IC₅₀ value of less than about60 pM as measured in an in vivo receptor/ligand binding bioassay at 25°C. The present invention also provides isolated antibodies, orantigen-binding fragments thereof, that specifically bind Activin A andblock activation of at least one Activin A receptor by Activin A. Insome embodiments of the invention, the isolated antibodies, orantigen-binding fragments thereof, do not significantly block binding ofActivin A to an Activin Type II receptor. In some embodiments of theinvention, the isolated antibodies, or antigen-binding fragmentsthereof, inhibit binding of Activin A to an Activin A receptor selectedfrom the group consisting of Activin Type IIA receptor (ActRIIA),Activin Type IIB receptor (ActRIIB), and Activin Type I receptor. Insome embodiments of the invention, the isolated antibodies, orantigen-binding fragments thereof, inhibit Activin A-mediated activationof SMAD complex signaling.

The present invention provides antibodies, or antigen-binding fragmentsthereof comprising a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 82, 98, 106, 114, 122, 130, 138, 154, 162, 170, 178, 186,194, and 202, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a light chain variable region (LCVR)having an amino acid sequence selected from the group consisting of SEQID NO: 10, 26, 42, 58, 74, 90, 146, and 210, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a HCVR and LCVR (HCVR/LCVR) sequence pairselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/90, 106/90, 114/90, 122/90, 130/90, 138/146,154/146, 162/146, 170/146, 178/146, 186/146, 194/146, and 202/210.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a heavy chain CDR3 (HCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 8, 24, 40, 56, 72, 88, 104, 112, 120, 128, 136, 144, 160, 168,176, 184, 192, 200, and 208, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; and a light chain CDR3 (LCDR3) domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 16, 32, 48,64, 80, 96, 152, and 216, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In certain embodiments, the antibody or antigen-binding portion of anantibody comprises a HCDR3/LCDR3 amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80,88/96, 104/96, 112/96, 120/96, 128/96, 136/96, 144/152, 160/152,168/152, 176/152, 184/152, 192/152, 200/152, and 208/216.

The present invention also provides an antibody or fragment thereoffurther comprising a heavy chain CDR1 (HCDR1) domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 20,36, 52, 68, 84, 100, 108, 116, 124, 132, 140, 156, 164, 172, 180, 188,196, and 204, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 110,118, 126, 134, 142, 158, 166, 174, 182, 190, 198, and 206, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a light chain CDR1(LCDR1) domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 148, and 212, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR2 (LCDR2) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14 (Trp Ala Ser SEQ ID NO: 14), 30 (AlaAla Ser SEQ ID NO: 30), 46 (Glu Thr Ser SEQ ID NO: 46), 62 (Asp Ala SerSEQ ID NO: 62), 78 (Asp Ala Ser SEQ ID NO: 78), 94(Ala Ala Ser SEQ IDNO: 94), 150 (Gly Ala Ser SEQ ID NO: 150), and 214 (Gly Ala Ser SEQ IDNO: 214), or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity.

Certain non-limiting, exemplary antibodies and antigen-binding fragmentsof the invention comprise HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences selected from the groupconsisting of: SEQ ID NOs: 4-6-8-12-14-16 (e.g. H4H10423P);20-22-24-28-30-32 (e.g. H4H10424P); 36-38-40-44-46-48 (e.g. H4H10426P);52-54-56-60-62-64 (e.g. H4H10429P); 68-70-72-76-78-80 (e.g. H4H10430P);84-86-88-92-94-96 (e.g. H4H10432P2); 100-102-104-92-94-96 (e.g.H4H10433P2); 108-110-112-92-94-96 (e.g. H4H10436P2);116-118-120-92-94-96 (e.g. H4H10437P2); 124-126-128-92-94-96 (e.g.H4H10438P2); 132-134-136-92-94-96 (e.g. H4H10440P2);140-142-144-148-150-152 (e.g. H4H10442P2); 156-158-160-148-150-152(H4H10445P2); 164-166-168-148-150-152 (H4H10446P2);172-174-176-148-150-152 (H4H10447P2); 180-182-184-148-150-152(H4H10448P2); 188-190-192-148-150-152 (H4H10452P2);196-198-200-148-150-152 (H4H10468P2); and 204-206-208-212-214-216(H2aM10965N).

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically binds ActivinA, wherein the antibody or fragment comprises the heavy and light chainCDR domains contained within heavy and light chain variable region(HCVR/LCVR) sequences selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/90, 106/90, 114/90, 122/90,130/90, 138/146, 154/146, 162/146,170/146, 178/146, 186/146,194/146, and202/210. Methods and techniques for identifying CDRs within HCVR andLCVR amino acid sequences are well known in the art and can be used toidentify CDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J Mol Biol 273:927-948 (1997); and Martin et al., PNAS (USA)86:9268-9272 (1989). Public databases are also available for identifyingCDR sequences within an antibody.

The present invention also provides nucleic acid molecules encodinganti-Activin A antibodies or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the HCVR aminoacid sequences listed in Table 1; in certain embodiments the nucleicacid molecule comprises a polynucleotide sequence selected from any ofthe HCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto. tgggcatct (SEQ ID NO: 13); gctgcatcc (SEQ ID NO: 29); gagacatcc(SEQ ID NO: 45); gatgcttcc (SEQ ID NO: 61); gatgcatcc (SEQ ID NO: 77);gctgcatcc (SEQ ID NO: 93); ggggcaagt (SEQ ID NO: 149); ggtgcatcc (SEQ IDNO: 213).

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-Activin A antibodieslisted in Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-Activin A antibodieslisted in Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-Activin A antibody listed inTable 1.

The present invention also provides recombinant expression vectorscapable of expressing a polypeptide comprising a heavy or light chainvariable region of an anti-Activin A antibody. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the HCVR, LCVR, and/or CDR sequences as set forth inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced.

The present invention includes anti-Activin A antibodies having amodified carbohydrate content. In some applications, modification toremove undesirable glycosylation sites may be useful. In someapplications, modification to alter glycosylation patterns may beuseful, e.g., modifying an antibody to lack a fucose moiety present onan oligosaccharide chain, for example, to increase antibody dependentcellular cytotoxicity (ADCC) function (see Shield et al. J Biol Chem277:26733 (2002)). In other applications, modification ofgalactosylation can be made in order to modify complement dependentcytotoxicity (CDC). In some applications, antibodies may have modifiedglycosylation patterns in order to minimize effector function. Forexample, antibodies may be modified to obtain additionally glycosylatedor sialylated antibodies.

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds Activin A and a pharmaceutically acceptable carrier.In a related aspect, the invention features a composition which is acombination of an anti-Activin A antibody and a second therapeuticagent. In one embodiment, the second therapeutic agent is any agent thatis advantageously combined with an anti-Activin A antibody. Exemplaryagents that may be advantageously combined with an anti-Activin Aantibody include, without limitation, other agents that inhibit ActivinA activity (including other antibodies or antigen-binding fragmentsthereof, peptide inhibitors, small molecule antagonists, etc.) and/oragents which do not directly bind Activin A but nonetheless interferewith, block or attenuate Activin A-mediated signaling. In oneembodiment, the secondary therapeutic agent inhibits, interferes, blocksand/or attenuates the activity of another ligand of the ActRIIA and/orActRIIB receptor (e.g., GDF8, Activin B, Activin AB, Inhibin A, InhibinB, GDF3, GDF11, Nodal, BMP2, BMP4, and/or BMP7). In one embodiment, thesecondary therapeutic agent is an anti-GDF8 antagonist (e.g., a humananti-GDF8 antibody or antigen-binding fragment thereof). Exemplaryanti-GDF8 agents for use with the anti-Activin A antibodies of theinvention include a human anti-GDF8 antibody (e.g., an anti-GDF8antibody comprising any of the HCVR/LCVR or CDR amino acid sequences asset forth in US 2011-0293630 A1 (e.g., H4H1657N2, which is an anti-GDF8antibody with heavy chain complementarity determining regions (HCDRs) ofa HCVR comprising SEQ ID NO:217 (e.g., the CDR1, CDR2, and CDR3sequences set forth in SEQ ID NO:218, 219, and 220, respectively), andthe light chain complementarity determining regions (LCDRs) of a LCVRcomprising SEQ ID NO:221 (e.g., the CDR1, CDR2, and CDR3 sequences setforth in SEQ ID NO:222, 223, and 224)(Thr Thr Ser SEQ ID NO: 223)).Additional combination therapies and co-formulations involving theanti-Activin A antibodies of the present invention are disclosedelsewhere herein.

In an additional aspect of the invention, an antigen-binding molecule isprovided comprising an Activin A-specific binding domain and aGDF8-specific binding domain. In one embodiment of this aspect of theinvention, the antigen-binding molecule is a bispecific antibodycomprising a first variable domain that specifically binds Activin A anda second variable domain that specifically binds GDF8.

In yet another aspect, the invention provides therapeutic methods forinhibiting Activin A activity using an anti-Activin A antibody orantigen-binding portion of an antibody of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising an antibody orantigen-binding fragment of an antibody of the invention. The disordertreated is any disease or condition which is improved, ameliorated,inhibited or prevented by removal, inhibition or reduction of Activin Aactivity or signaling. The anti-Activin A antibodies or antibodyfragments of the invention may function to block the interaction betweenActivin A and an Activin Type II receptor (e.g., Activin Type IIAreceptor and/or Activin Type IIB receptor); between Activin A and anActivin Type I receptor; between Activin A and both a Type II and a TypeI receptor; or otherwise inhibit the signaling activity of Activin A.

The present invention also includes the use of an anti-Activin Aantibody or antigen binding portion of an antibody of the invention inthe manufacture of a medicament for the treatment of a disease ordisorder related to or caused by Activin A activity in a patient. Thepresent invention also provides methods for increasing muscle mass orstrength in a subject by administering to the subject an Activin Aantibody or antigen-binding fragment thereof. The present invention alsoprovides methods for increasing muscle mass or strength in a subject byadministering to the subject an Activin A-specific binding protein and aGDF8-specific binding protein, or by administering to the subject anantigen-binding molecule comprising an Activin A-specific binding domainand a GDF8-specific binding domain.

The invention also includes methods for treating, preventing and/orameliorating a disease or disorder characterized by decreased musclemass or strength by administering to a subject in need thereof anActivin A-specific binding protein (e.g., an anti-Activin A antibody).In a related aspect, methods of the invention include the treating,preventing and/or ameliorating a disease or disorder characterized bydecreased muscle mass or strength by administering to a subject in needthereof an Activin A-specific binding protein and a GDF8-specificbinding protein (e.g., an anti-Activin A antibody and an anti GDF8antibody). Methods of the invention also include treating, preventingand/or ameliorating a disease or disorder characterized by decreasedmuscle mass or strength by administering to a subject in need thereof anantigen-binding molecule comprising an Activin A-specific binding domainand a GDF8-specific binding domain. Diseases or disorders characterizedby decreased muscle mass or strength that can be treated, preventedand/or ameliorated using methods of the invention include sarcopenia,cachexia (e.g., idiopathic cachexia or cachexia secondary to anothercondition (e.g., cancer, chronic renal failure, or chronic obstructivepulmonary disease)), muscle injury, muscle wasting and/or atrophy (e.g.,caused by or associated with disuse, immobilization, bed rest, injury,medical treatment, surgical intervention (e.g., hip fracture, hipreplacement, and knee replacement) and by necessity of mechanicalventilation), cancer, obesity, diabetes, arthritis, multiple sclerosis,muscular dystrophy, amyotrophic lateral sclerosis, Parkinson’s disease,osteoporosis, osteoarthritis, osteopenia, and metabolic syndromes (e.g.,one or more of diabetes, obesity, nutritional disorders, organ atrophy,chronic obstructive pulmonary disease, and anorexia).

The invention also includes methods for treating, preventing and/orameliorating diseases or disorders caused by, promoted by, exacerbatedby, or aggravated by the activity of a molecule containing inhibin βA(e.g., dimers containing inhibin βA, e.g., Activin A, Activin AB, etc.)by administering to a subject in need thereof a binding protein specificfor Activin A (i.e., inhibin βA dimer), e.g., an anti-Activin A antibodyor antigen-binding fragment thereof. In one aspect of the invention,methods of the invention include methods of treating, preventing, and/orameliorating renal fibrosis by administering to a subject in needthereof an anti-Activin A antibody. In particular aspects of theinvention, methods of the invention include methods of treating,preventing, and/or ameliorating renal fibrosis caused by chronic kidneydisease (e.g., as a consequence of hypertension, diabetes,glomerulonephritis, inherited diseases (such as polycystic kidneydisease), malformations of the kidney, autoimmune disease (e.g., lupus),or obstructions (e.g., kidney stones, tumors, enlarged prostate gland),or repeated urinary infections) by administering to a subject in needthereof an anti-Activin A antibody. Additional aspects of the inventioninclude methods of treating, preventing, and/or ameliorating sepsis,chronic heart failure, chronic obstructive pulmonary disease, benign ormalignant pheochromocytoma, uterine fibroids/leiomyomata, preeclampsia,keloids, hypertrophic scars, or pulmonary artery hypertension byadministering to a subject in need thereof an anti-Activin A antibody.Additional aspects of the invention include methods of treating,preventing, and/or ameliorating cachexia caused by, promoted by,exacerbated by,or aggravated by Activin A activity by administering to asubject in need thereof an anti-Activin A antibody. Additional aspectsof the invention include methods of treating, preventing, and/orameliorating weight loss caused by, promoted by, exacerbated by,oraggravated by Activin A activity by administering to a subject in needthereof an anti-Activin A antibody.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a matrix showing the results of an antibody cross-competitionassay in which a first anti-Activin A antibody (“Antibody Sample”) wasapplied to an anti-human FC-coated sensor tip, followed by emersion in asolution of a second anti-Activin A antibody (1 µM) pre-bound to ActivinA. Binding responses (numerical values 0.22 to 1.84) for each antibodycombination tested are depicted. Binding responses presented in whiteboxes with black type indicate no competition for binding of Activin A,suggesting distinct binding regions.

FIG. 2 : Panel A shows the effects of 21 days of anti-GDF8 antibodytreatment (H4H1657N2, 10 mg/kg or 30 mg/kg) on average peak tetanicforce compared to isotype control antibody. Data analyzed using one- wayanalysis of variance (ANOVA) followed by Tukey’s test. *p<0.05significance over isotype control (n=6, unpaired Student t test); n.s. =not statistically significant compared to isotype 30 mg/kg. Panel Bshows the increase in tibialis anterior (TA) muscle peak tetanic forcein H4H1657N2-treated mice (10 mg/kg) versus mice treated with isotypecontrol antibodies for three weeks (n=6), when stimulated by electriccurrent over a range of frequencies (40 to 100 Hz). Data are expressedas mean average peak force ± SEM.

FIG. 3 : Panel A shows the design of an experiment to evaluate theeffects of H4H1657N2 during the recovery phase from hind limbsuspension-induced muscle atrophy. Panel B shows the percentage changein TA and Gastrocnemius (GA) muscle weights for H4H1657N2-treated andisotyple control antibody-treated mice post-recovery after 7 days ofhind limb suspension (HLS+7Rec) versus mice without a recovery periodafter 7 days of hind limb suspension (HLS) and control mice (non-HLScontrol). Values are expressed as the mean percentage change overcontrol non-HLS values ± SEM. Data analyzed using one- way analysis ofvariance (ANOVA) followed by Tukey’s test. * = p<0.05 significance overNon-HLS group. # = p<0.05 significance over HLS group.

FIG. 4 : Panel A shows the effects of the administration of theanti-Activin A antibody H4H10446P2 on body weight of mice overexpressingActivin A (versus isotype control). Data was analyzed using two-wayanalysis of variance (Repeated Measures ANOVA + Boneferroni MultipleComparison Test) followed by Tukey’s test. *= p<0.05 vs Isotype Control;#= p<0.05 vs Activin A + Isotype Control. Panel B shows the effects ofanti-Activin A antibody H4H10446P2 on tibialis anterior (TA) andGastrocnemius (GA) muscle weights in mice overexpressing Activin A(versus isotype control). Data analyzed using one- way analysis ofvariance (ANOVA) followed by Tukey’s test. *= p<0.05 over Vector +Isotype Control; #= p<0.05 over Activin A + Isotype Control.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Antigen-Specific Binding Proteins

The present invention relates to compositions comprisingantigen-specific binding proteins. More specifically, the presentinvention provides a composition comprising an Activin A-specificbinding protein.

As used herein, the expression “antigen-specific binding protein” meansa protein comprising at least one domain which specifically binds aparticular antigen. Exemplary categories of antigen-specific bindingproteins include antibodies, antigen-binding portions of antibodies,peptides that specifically interact with a particular antigen (e.g.,peptibodies), receptor molecules that specifically interact with aparticular antigen, and proteins comprising a ligand-binding portion ofa receptor that specifically binds a particular antigen.

The present invention includes antigen-specific binding proteins thatspecifically bind Activin A, i.e., “Activin A-specific bindingproteins”. Activins are homo- and hetero-dimeric molecules comprisingbeta subunits, i.e., Inhibin βA, inhibin βB, inhibin βC, and/or inhibinβE. The βA subunit has the amino acid sequence of SEQ ID NO:226 and theβB subunit has the amino acid sequence of SEQ ID NO:228. Activin A is ahomodimer of two βA subunits; Activin B is a homodimer of two βBsubunits; Activin AB is a heterodimer of one βA subunit and one βBsubunit; and Activin AC is a heterodimer of one βA subunit and one βCsubunit. An Activin A-specific binding protein may be anantigen-specific binding protein that specifically binds the βA subunit.Since the βA subunit is found in Activin A, Activin AB, and Activin ACmolecules, an “Activin A-specific binding protein” can be anantigen-specific binding protein that specifically binds Activin A aswell as Activin AB and Activin AC (by virtue of its interaction with theβA subunit). Therefore, according to one embodiment of the presentinvention, an Activin A-specific binding protein specifically bindsActivin A; or Activin A and Activin AB; or Activin A and Activin AC; orActivin A, Activin AB and Activin AC, but does not bind other ActRIIBligands such as Activin B, GDF3, GDF8, BMP2, BMP4, BMP7, BMP9, BMP10,GDF11, Nodal, etc. Thus, in one embodiment of the invention, an ActivinA-specific binding protein specifically binds to Activin A but does notbind significantly to Activin B or Activin C. In another embodiment, anActivin A-specific binding protein may also bind to Activin B (by virtueof cross-reaction with the βB subunit, i.e., InhibinβB). In anotherembodiment, an Activin A-specific binding protein is a binding proteinthat binds specifically to Activin A but does not bind to any otherligand of ActRIIB. In another embodiment, an Activin A-specific bindingprotein is a binding protein and binds specifically to Activin A anddoes not bind to any Bone Morphogenetic Protein (BMP) (e.g., BMP2, BMP4,BMP6, BMP9, BMP10). In another embodiment, an Activin A-specific bindingprotein is a binding protein that binds specifically to Activin A butdoes not bind to any other member of the transforming growth factor beta(TGFβ) superfamily.

The present invention also includes antigen-specific binding proteinsthat specifically bind GDF8, i.e., “GDF8-specific binding proteins”. Theterm “GDF8” (also referred to as “growth and differentiation factor-8”and “myostatin”) means the protein having the amino acid sequence of SEQID NO:225 (mature protein). According to the present invention,GDF8-specific binding proteins specifically bind GDF8 but do not bindother ActRIIB ligands such as GDF3, BMP2, BMP4, BMP7, BMP9, BMP10,GDF11, Activin A, Activin B, Activin AB, Nodal, etc.

In the context of the present invention, molecules such as ActRIIB-Fc(e.g., “ACE-031”), which comprise the ligand-binding portion of theActRIIB receptor, are not considered “Activin A-specific bindingproteins” or “GDF8-specific binding proteins” because such moleculesbind multiple ligands besides GDF8, Activin A and Activin AB.

All references to proteins, polypeptides and protein fragments hereinare intended to refer to the human version of the respective protein,polypeptide or protein fragment unless explicitly specified as beingfrom a non-human species.

Antigen-Binding Molecules With Two Different Antigen-Specific BindingDomains

The present invention also includes antigen-binding molecules comprisingtwo different antigen-specific binding domains. In particular, thepresent invention includes antigen-binding molecules comprising anActivin A-specific binding domain and a GDF8-specific binding domain.The term “antigen-specific binding domain,” as used herein, includespolypeptides comprising or consisting of: (i) an antigen-bindingfragment of an antibody molecule, (ii) a peptide that specificallyinteracts with a particular antigen (e.g., a peptibody), and/or (iii) aligand-binding portion of a receptor that specifically binds aparticular antigen. For example, the present invention includesbispecific antibodies with one arm comprising a first heavy chainvariable region/light chain variable region (HCVR/LCVR) pair thatspecifically binds Activin A and another arm comprising a secondHCVR/LCVR pair that specifically binds GDF8.

Specific Binding

The term “specifically binds” or the like, as used herein, means that anantigen-specific binding protein, or an antigen-specific binding domain,forms a complex with a particular antigen characterized by adissociation constant (K_(D)) of 500 pM or less, and does not bind otherunrelated antigens under ordinary test conditions. “Unrelated antigens”are proteins, peptides or polypeptides that have less than 95% aminoacid identity to one another. Methods for determining whether twomolecules specifically bind one another are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. For example, an antigen-specific binding protein or anantigen-specific binding domain, as used in the context of the presentinvention, includes molecules that bind a particular antigen (e.g.,Activin A and/or AB, or GDF8) or a portion thereof with a K_(D) of lessthan about 500 pM, less than about 400 pM, less than about 300 pM, lessthan about 200 pM, less than about 100 pM, less than about 90 pM, lessthan about 80 pM, less than about 70 pM, less than about 60 pM, lessthan about 50 pM, less than about 40 pM, less than about 30 pM, lessthan about 20 pM, less than about 10 pM, less than about 5 pM, less thanabout 4 pM, less than about 2 pM, less than about 1 pM, less than about0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less thanabout 0.05 pM, as measured in a surface plasmon resonance assay.

As used herein, an antigen-specific binding protein or antigen-specificbinding domain “does not bind” to a specified molecule (e.g., “does notbind GDF11”, “does not bind BMP9”, “does not bind BMP10”, etc.) if theprotein or binding domain, when tested for binding to the molecule at25° C. in a surface plasmon resonance assay, exhibits a K_(D) of greaterthan 50.0 nM, or fails to exhibit any binding in such an assay orequivalent thereof.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

The term “K_(D) ” as used herein, means the equilibrium dissociationconstant of a particular protein-protein interaction (e.g.,antibody-antigen interaction). Unless indicated otherwise, the K_(D)values disclosed herein refer to K_(D) values determined by surfaceplasmon resonance assay at 25° C.

Antibodies and Antigen-Binding Fragments of Antibodies

As indicated above, an antigen-specific binding protein can comprise orconsist of an antibody or antigen-binding fragment of an antibody.Furthermore, in the case of antigen-binding molecules comprising twodifferent antigen-specific binding domains, one or both of theantigen-specific binding domains may comprise or consist of anantigen-binding fragment of an antibody.

As used herein, “an antibody that binds Activin” or an “anti-Activin Aantibody” includes antibodies, and antigen-binding fragments thereof,that bind a soluble fragment of the Activin A protein and may also bindto an Activin βA subunit-containing Activin heterodimer.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., Activin A). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-Activin A antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al., PNAS USA 95:652-656 (1998)). The constantregion of an antibody is important in the ability of an antibody to fixcomplement and mediate cell-dependent cytotoxicity. Thus, the isotype ofan antibody may be selected on the basis of whether it is desirable forthe antibody to mediate cytotoxicity.

In certain embodiments of the invention, the anti-Activin A antibodiesof the invention are human antibodies. The term “human antibody”, asused herein, is intended to include antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al., Nucl Acids Res20:6287-6295 (1992)) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.Molecular Immunology 30:105 1993)) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The present invention includes neutralizing and/or blocking anti-ActivinA antibodies. A “neutralizing” or “blocking” antibody, as used herein,is intended to refer to an antibody whose binding to Activin A: (i)interferes with the interaction between Activin A and an Activin Areceptor (e.g., Activin Type IIA receptor, Activin Type IIB receptor,Activin Type I receptor, etc.); (ii) interferes with the formation ofActivin-Activin receptor complexes; and/or (iii) results in inhibitionof at least one biological function of Activin A. The inhibition causedby an Activin A neutralizing or blocking antibody need not be completeso long as it is detectable using an appropriate assay. Exemplary assaysfor detecting Activin A inhibition are described in the working Examplesherein.

The anti-Activin A antibodies disclosed herein may comprise one or moreamino acid substitutions, insertions and/or deletions in the frameworkand/or CDR regions of the heavy and light chain variable domains ascompared to the corresponding germline sequences from which theantibodies were derived. Such mutations can be readily ascertained bycomparing the amino acid sequences disclosed herein to germlinesequences available from, for example, public antibody sequencedatabases. The present invention includes antibodies, andantigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-Activin A antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-Activin A antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson, W.R.,Methods Mol Biol 24: 307-331 (1994), herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-1445 (1992), herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(see, e.g., Pearson, W.R., Methods Mol Biol 132: 185-219 (2000), hereinincorporated by reference). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al., J Mol Biol 215:403-410 (1990) and Altschul et al.,Nucleic Acids Res 25:3389-402 (1997), each herein incorporated byreference.

Biological Characteristics of the Antibodies

The present invention includes anti-Activin A antibodies andantigen-binding fragments thereof that bind Activin A with highaffinity. For example, the present invention includes antibodies andantigen-binding fragments of antibodies that bind Activin A (e.g., at25° C. or 37° C.) with a K_(D) of less than about 30 nM as measured bysurface plasmon resonance, e.g., using the assay format as defined inExample 3 herein. In certain embodiments, the antibodies orantigen-binding fragments of the present invention bind Activin A with aK_(D) of less than about 25 nM, less than about 20 nM, less than about15 nM, less than about 10 nM, less than about 5 nM, less than about 2nM, less than about 1 nM, less than about 500 pM, less than about 250pM, less than about 240 pM, less than about 230 pM, less than about 220pM, less than about 210 pM, less than about 200 pM, less than about 190pM, less than about 180 pM, less than about 170 pM, less than about 160pM, less than about 150 pM, less than about 140 pM, less than about 130pM, less than about 120 pM, less than about 110 pM, less than about 100pM, less than about 95 pM, less than about 90 pM, less than about 85 pM,less than about 80 pM, less than about 75 pM, less than about 70 pM,less than about 65 pM, less than about 60 pM, less than about 55 pM,less than about 50 pM, less than about 45 pM, less than about 40 pM,less than about 35 pM, less than about 30 pM, less than about 25 pM,less than about 20 pM, less than about 15 pM, less than about 10 pM,less than about 9 pM, less than about 8 pM, less than about 7 pM, lessthan about 6 pM, less than about 5 pM, less than about 4 pM, or lessthan about 3 pM, as measured by surface plasmon resonance, e.g., usingthe assay format as defined in Example 3 herein, or a substantiallysimilar assay.

The present invention also includes anti-Activin A antibodies andantigen-binding fragments thereof that inhibit Activin A-mediatedcellular signaling. For example, the present invention includesanti-Activin A antibodies that inhibit the activation of the SMADcomplex signal transduction pathway via the binding of Activin A toActivin Type I or II receptors with an IC₅₀ value of less than about 4nM, as measured in a cell-based blocking bioassay, e.g., using the assayformat as defined in Example 6 herein, or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention inhibit the activation of the SMAD complex signaltransduction pathway via the binding of Activin A to Activin Type I orII receptors with an IC₅₀ value of less than about 3 nM, less than about2 nM, less than about 1 nm, less than about 500 pM, less than about 250pM, less than about 240 pM, less than about 230 pM, less than about 220pM, less than about 210 pM, less than about 200 pM, less than about 190pM, less than about 180 pM, less than about 170 pM, less than about 160pM, less than about 150 pM, less than about 140 pM, less than about 130pM, less than about 120 pM, less than about 110 pM, less than about 100pM, less than about 95 pM, less than about 90 pM, less than about 85 pM,less than about 80 pM, less than about 75 pM, less than 70 pM, less thanabout 65 pM, less than about 60 pM, less than about 55 pM, less thanabout 50 pM, less than about 49 pM, less than about 48 pM, less thanabout 47 pM, less than about 46 pM, less than about 45 pM, less thanabout 44 pM, less than about 43 pM, less than about 42 pM, less thanabout 41 pM, less than about 40 pM, or less than about 39 pM, asmeasured in a cell-based blocking bioassay, e.g., using the assay formatas defined in Example 6 herein, or a substantially similar assay. Incertain embodiments, the antibodies or antigen-binding fragments of thepresent invention inhibit the signaling activing of Activin B byinterfering with the binding of Activin B to Activin Type I or IIreceptors with an IC₅₀ value of less than about 50 nM, less than about20 nM, less than about 10 nm, less than about 5 nM, or less than about 1nM, as measured in a cell-based blocking bioassay, e.g., using the assayformat as defined in Example 6 herein, or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention inhibit the activation of the SMAD complex signaltransduction pathway via the binding of Activin AB to Activin Type I orII receptors with an IC₅₀ value of less than about 500 pM, less thanabout 450 pM, less than about 440 pM, less than about 430 pM, less thanabout 420 pM, less than about 410 pM, less than about 400 pM, less thanabout 390 pM, less than about 380 pM, less than about 370 pM, less thanabout 360 pM, less than about 350 pM, less than about 340 pM, less thanabout 320 pM, less than about 310 pM, less than about 300 pM, less thanabout 290 pM, less than about 280 pM, less than about 270 pM, less thanabout 260 pM, less than about 250 pM, less than about 240 pM, less thanabout 230 pM, less than about 220 pM, less than about 210 pM, less thanabout 200 pM, less than about 190 pM, less than about 180 pM, less thanabout 170 pM, less than about 160 pM, less than about 150 pM, or lessthan about 140 pM, as measured in a cell-based blocking bioassay, e.g.,using the assay format as defined in Example 6 herein, or asubstantially similar assay. In certain embodiments, the antibodies orantigen-binding fragments of the present invention inhibit theactivation of the SMAD complex signal transduction pathway via thebinding of Activin AC to Activin Type I or II receptors with an IC₅₀value of less than about 1 nM, less than about 900 pM, less than about800 pM, less than about 750 pM, less than about 700 pM, less than about650 pM, less than about 600 pM, or less than about 580 pM, as measuredin a cell-based blocking bioassay, e.g., using the assay format asdefined in Example 6 herein, or a substantially similar assay.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combinations thereof.Other biological characteristics of the antibodies of the presentinvention will be evident to a person of ordinary skill in the art froma review of the present disclosure including the working Examplesherein.

Anti-Activin A Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-ActivinA antibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-Activin A antibodies comprising amutation in the C_(H)2 or a C_(H)3 region of the Fc domain, wherein themutation(s) increases the affinity of the Fc domain to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). Such mutations may result in an increase in serum half-lifeof the antibody when administered to an animal. Non-limiting examples ofsuch Fc modifications include, e.g., a modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification atposition 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W,H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). Inyet another embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-Activin A antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I andQ311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g.,D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A);and 433K and 434F (e.g., H433K and N434F). All possible combinations ofthe foregoing Fc domain mutations, and other mutations within theantibody variable domains disclosed herein, are contemplated within thescope of the present invention.

The present invention also includes anti-Activin A antibodies comprisinga chimeric heavy chain constant (CH) region, wherein the chimeric CHregion comprises segments derived from the CH regions of more than oneimmunoglobulin isotype. For example, the antibodies of the invention maycomprise a chimeric CH region comprising part or all of a CH2 domainderived from a human IgG1, human IgG2 or human IgG4 molecule, combinedwith part or all of a CH3 domain derived from a human IgG1, human IgG2or human IgG4 molecule. According to certain embodiments, the antibodiesof the invention comprise a chimeric CH region having a chimeric hingeregion. For example, a chimeric hinge may comprise an “upper hinge”amino acid sequence (amino acid residues from positions 216 to 227according to EU numbering) derived from a human IgG1, a human IgG2 or ahuman IgG4 hinge region, combined with a “lower hinge” sequence (aminoacid residues from positions 228 to 236 according to EU numbering)derived from a human IgG1, a human IgG2 or a human IgG4 hinge region.According to certain embodiments, the chimeric hinge region comprisesamino acid residues derived from a human IgG1 or a human IgG4 upperhinge and amino acid residues derived from a human IgG2 lower hinge. Anantibody comprising a chimeric CH region as described herein may, incertain embodiments, exhibit modified Fc effector functions withoutadversely affecting the therapeutic or pharmacokinetic properties of theantibody. (See, e.g., U.S. Provisional Appl. No. 61/759,578, filed Feb.1, 2013, the disclosure of which is hereby incorporated by reference inits entirety).

Epitope Mapping and Related Technologies

The present invention includes anti-Activin A antibodies which interactwith one or more amino acids found within Activin A (e.g., within theActivin Type II receptor binding site). The epitope to which theantibodies bind may consist of a single contiguous sequence of 3 or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20or more) amino acids located within the Activin βA subunit.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) located within the Activin Adimer.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY),alanine scanning mutational analysis, peptide blots analysis (Reineke,Methods Mol Biol 248:443-463 (2004)), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, ProteinScience 9:487-496 (2000)). Another method that can be used to identifythe amino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring, Analytical Biochemistry 267(2):252-259(1999); Engen and Smith, Anal. Chem. 73:256A-265A (2001).

The present invention further includes anti-Activin A antibodies thatbind to the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g., H4H10423P, H4H10424P, H4H10426P, H4H10429P,H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2,H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10448P2,H4H10452P2, H4H10468P2, H2aM10965N, etc.). Likewise, the presentinvention also includes anti-Activin A antibodies that compete forbinding to Activin A with any of the specific exemplary antibodiesdescribed herein (e.g., H4H10423P, H4H10424P, H4H10426P, H4H10429P,H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2,H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10448P2,H4H10452P2, H4H10468P2, H2aM10965N, etc.). For example, the presentinvention includes anti-Activin A antibodies that cross-compete forbinding to Activin A with one or more antibodies of “Bin 1” as definedin Example 5 herein (e.g., H4H10423P, H4H10446P2, H4H10468P2 andH4H10442P2). The present invention also includes anti-Activin Aantibodies that cross-compete for binding to Activin A with one or moreantibodies of “Bin 2” as defined in Example 5 herein (e.g., H4H10429,H4H10430P, H4H10432P2, H4H10436P2, and H4H10440P2).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-Activin A antibody byusing routine methods known in the art and exemplified herein. Forexample, to determine if a test antibody binds to the same epitope as areference anti-Activin A antibody of the invention, the referenceantibody is allowed to bind to Activin A (or a βA subunit-containingheterodimer). Next, the ability of a test antibody to bind to Activin Ais assessed. If the test antibody is able to bind to Activin A followingsaturation binding with the reference anti-Activin A antibody, it can beconcluded that the test antibody binds to a different epitope than thereference anti-Activin A antibody. On the other hand, if the testantibody is not able to bind to Activin A following saturation bindingwith the reference anti-Activin A antibody, then the test antibody maybind to the same epitope as the epitope bound by the referenceanti-Activin A antibody of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, BIACORE™ real-time surface plasmonresonance biosensor flow cytometry or any other quantitative orqualitative antibody-binding assay available in the art. In accordancewith certain embodiments of the present invention, two antibodies bindto the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or100-fold excess of one antibody inhibits binding of the other by atleast 50% but preferably 75%, 90% or even 99% as measured in acompetitive binding assay (see, e.g., Junghans et al., Cancer Res.50:1495-1502 (1990)). Alternatively, two antibodies are deemed to bindto the same epitope if essentially all amino acid mutations in theantigen that reduce or eliminate binding of one antibody reduce oreliminate binding of the other. Two antibodies are deemed to have“overlapping epitopes” if only a subset of the amino acid mutations thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-Activin A antibody, the above-describedbinding methodology is performed in two orientations: In a firstorientation, the reference antibody is allowed to bind to Activin Aprotein (or a βA subunit-containing heterodimer) under saturatingconditions followed by assessment of binding of the test antibody to theActivin A molecule. In a second orientation, the test antibody isallowed to bind to Activin A under saturating conditions followed byassessment of binding of the reference antibody to Activin A. If, inboth orientations, only the first (saturating) antibody is capable ofbinding to Activin A, then it is concluded that the test antibody andthe reference antibody compete for binding to Activin A (see, e.g., theassay format described in Example 4 herein, in which a test Activin Aantibody is captured onto sensor tips that are then submerged in asolution containing a reference Activin A antibody pre-bound withActivin A). As will be appreciated by a person of ordinary skill in theart, an antibody that competes for binding with a reference antibody maynot necessarily bind to the same epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Anti-Activin A antibodies of the invention may bind to an epitope onActivin A that is within or near the binding site for an Activin Type IIreceptor, directly block interaction between Activin A and an ActivinType II receptor, and indirectly block interaction between Activin A andan Activin Type I receptor. Anti-Activin A antibodies of the inventionmay bind to an epitope on Activin A that is within or near the bindingsite for the Activin Type I receptor and directly block interactionbetween Activin A and an Activin Type I receptor. In one embodiment ofthe invention, an anti-Activin A antibody of the invention that binds toActivin A at or near the Activin Type I receptor binding site does notblock interaction between Activin A and an Activin A Type II receptor.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human Activin A.

Using VELOCIMMUNE™ technology, for example, or any other known methodfor generating fully human monoclonal antibodies, high affinity chimericantibodies to human Activin A are initially isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. If necessary, mouse constant regions are replaced with a desiredhuman constant region, for example wild-type or modified IgG1 or IgG4,to generate a fully human anti-Activin A antibody. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region. In certain instances, fully human anti-Activin Aantibodies are isolated directly from antigen-positive B cells.

Bioequivalents

The anti-Activin A antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies but that retain the ability to bindhuman Activin A. Such variant antibodies and antibody fragments compriseone or more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the anti-Activin A antibody-encoding DNA sequences of the presentinvention encompass sequences that comprise one or more additions,deletions, or substitutions of nucleotides when compared to thedisclosed sequence, but that encode an anti-Activin A antibody orantibody fragment that is essentially bioequivalent to an anti-Activin Aantibody or antibody fragment of the invention. Examples of such variantamino acid and DNA sequences are discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-Activin A antibodies of the invention maybe constructed by, for example, making various substitutions of residuesor sequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-Activin A antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention, according to certain embodiments, providesanti-Activin A antibodies that bind to human Activin A but not toActivin A from other species. The present invention also includesanti-Activin A antibodies that bind to human Activin A and to Activin Afrom one or more non-human species. For example, the anti-Activin Aantibodies of the invention may bind to human Activin A and may bind ornot bind, as the case may be, to one or more of mouse, rat, guinea pig,hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel,cynomologous, marmoset, rhesus or chimpanzee Activin A. According tocertain exemplary embodiments of the present invention, anti-Activin Aantibodies are provided which specifically bind human Activin A (e.g.,Activin A or a βA subunit-containing heterodimer) and cynomolgus monkey(e.g., Macaca fascicularis) Activin A.

Immunoconjugates

The invention encompasses anti-Activin A monoclonal antibodiesconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxic agents include any agent that is detrimental tocells. Examples of suitable cytotoxic agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art (see for example, WO05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., J Immunol 147:60-69 (1991); Kufer et al., TrendsBiotechnol 22:238-244 (2004). The anti-Activin A antibodies of thepresent invention can be linked to or co-expressed with anotherfunctional molecule, e.g., another peptide or protein. For example, anantibody or fragment thereof can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody orantibody fragment to produce a bi-specific or a multispecific antibodywith a second binding specificity. For example, the present inventionincludes bi-specific antibodies wherein one arm of an immunoglobulin isspecific for human Activin A or a fragment thereof, and the other arm ofthe immunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety. One embodiment of the inventionincludes bi-specific antibodies wherein one arm of an immunoglobulin isspecific for human Activin A or a fragment thereof, and the other arm ofthe immunoglobulin is specific for GDF8.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference (see, e.g., US Pat. No.8,586,713, incorporated by reference herein in its entirety). In oneembodiment, the first Ig C_(H)3 domain binds Protein A and the second IgC_(H)3 domain contains a mutation that reduces or abolishes Protein Abinding such as an H95R modification (by IMGT exon numbering; H435R byEU numbering). The second C_(H)3 may further comprise a Y96Fmodification (by IMGT; Y436F by EU). Further modifications that may befound within the second C_(H)3 include: D16E, L18M, N44S, K52N, V57M,and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU)in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S,K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S,K52N, V57M, R69K, E79Q, V82I, and L105P (by IMGT; Q355R, N384S, K392N,V397M, R409K, E419Q, V422I, and L445P by EU) in the case of IgG4antibodies. Variations on the bi-specific antibody format describedabove are contemplated within the scope of the present invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al., mAbs4:6, 1-11 (2012), and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J Am Chem Soc. 135(1):340-346 (2013)).

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-Activin A antibodies or antigen-binding fragments thereof of thepresent invention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington’s PharmaceuticalSciences, Mack Publishing Company, Easton, PA. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing CARBOWAX™. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA, J PharmSci Technol 52:238-311 (1998).

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with Activin A activity in an adult patient, it maybe advantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering anti-ActivinA antibodies may be determined empirically; for example, patientprogress can be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., PharmaceutRes 8:1351 (1991)).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing an antibody or other therapeutic protein of the invention,receptor mediated endocytosis (see, e.g., Wu et al., J Biol Chem262:4429-4432 (1987)). The antibodies and other therapeutically activecomponents of the present invention may also be delivered by genetherapy techniques. Methods of introduction include, but are not limitedto, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The composition maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPENJUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson,Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™ OPTIPEN STARLET™, andOPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few.Examples of disposable pen delivery devices having applications insubcutaneous delivery of a pharmaceutical composition of the presentinvention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN® (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987)).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Florida. In yet another embodiment, a controlledrelease system can be placed in proximity of the composition’s target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, Science 249:1527-1533 (1990).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by Activin A expression, signaling, oractivity, or treatable by blocking the interaction between Activin A andan Activin A receptor (e.g., ActRIIA, ActRIIB, Activin Type I receptor,etc.) or otherwise inhibiting Activin A activity and/or signaling. Forexample, the present invention provides methods of treating conditionsor afflictions which can be cured, alleviated or improved by increasingmuscle strength/power and/or muscle mass and/or muscle function in anindividual, or by favorably altering metabolism (carbohydrate, lipid andprotein processing) by specifically binding Activin A and not bindingother ActRIIB ligands, or by specifically binding Activin A and GDF8 andnot binding other ActRIIB ligands. For example, the present inventionincludes methods for increasing muscle strength/power and/or muscle massand/or muscle function in a subject, or for treating a disease ordisorder characterized by decreased muscle mass or strength in asubject, by administering to the subject an Activin A-specific bindingprotein. The present invention also includes methods for increasingmuscle strength/power and/or muscle mass and/or muscle function in asubject, or for treating a disease or disorder characterized bydecreased muscle mass or strength in a subject, by administering to thesubject an Activin A-specific binding protein and a GDF8-specificbinding protein. Any of the Activin A-specific binding proteins and/orGDF8-specific binding proteins disclosed or referred to herein can beused in the context of these aspects of the invention. For example, thetherapeutic methods of the present invention include administering to asubject an anti-Activin A antibody and/or an anti-GDF8 antibody.

Thus, in the context of the methods of treatment described herein, theanti-Activin A antibody may be administered as a monotherapy (i.e., asthe only therapeutic agent) or in combination with one or moreadditional therapeutic agents (e.g., an anti-GDF8 antibody), furtherexamples of which are described elsewhere herein.

In methods which comprise administering an Activin A-specific bindingprotein and a GDF8-specific binding protein to a subject, the ActivinA-specific binding protein and the GDF8-specific binding protein may beadministered to the subject at the same or substantially the same time,e.g., in a single therapeutic dosage, or in two separate dosages whichare administered simultaneously or within less than about 5 minutes ofone another. Alternatively, the Activin A-specific binding protein andthe GDF8-specific binding protein may be administered to the subjectsequentially, e.g., in separate therapeutic dosages separated in timefrom one another by more than about 5 minutes.

The present invention also includes methods for increasing musclestrength/power and/or muscle mass and/or muscle function in a subject,or for treating a disease or disorder characterized by decreased musclemass or strength in a subject, by administering to the subject anantigen-binding molecule comprising an Activin A-specific binding domainand a GDF8-specific binding domain. Any of the antigen-binding moleculesdisclosed or referred to herein can be used in the context of thisaspect of the invention. For example, the therapeutic methods of thepresent invention include administering to a subject a bispecificantibody comprising a first variable domain comprising a HCVR/LCVR pairthat specifically binds Activin A and a second variable domaincomprising a HCVR/LCVR pair that specifically binds GDF8.

The compositions of the present invention may be administered to asubject along with one or more additional therapeutic agents, including,e.g., growth factor inhibitors, immunosuppressants, anti-inflammatoryagents, metabolic inhibitors, enzyme inhibitors, andcytotoxic/cytostatic agents. The additional therapeutic agent(s) may beadministered prior to, concurrent with, or after the administration ofthe Activin A- and GDF8-specific binding proteins of the presentinvention.

Exemplary diseases, disorders and conditions that can be treated withthe compositions of the present invention include, but are not limitedto, sarcopenia, cachexia (either idiopathic or secondary to otherconditions, e.g., cancer, chronic renal failure, or chronic obstructivepulmonary disease), muscle injury, muscle trauma, muscle wasting andmuscle atrophy, e.g., muscle atrophy or wasting caused by or associatedwith disuse, e.g., muscular, immobilization, bed rest, injury, medicaltreatment or surgical intervention (e.g., hip fracture, hip replacement,knee replacement, and other joint, tendon, or ligament injuries such astears in the anterior cruciate ligament (ACL) and/or the medialcollateral ligament (MCL), etc.), muscular dystrophy (e.g., Myotonic,Duchenne, Becker, Limb-girdle, Facioscapulohumeral (FSHD, also known asLandouzy-Déjérine disease), Congenital, Oculopharyngeal, Distal,Emery-Dreifuss, etc.), glucocorticoid-induced myopathy, strokerehabilitation (e.g., rehabilitation for stroke hemiparesis) or bynecessity of mechanical ventilation. The compositions of the inventionmay also be used to treat, prevent or ameliorate diseases such ascancer, obesity, diabetes, arthritis, multiple sclerosis, musculardystrophy, amyotrophic lateral sclerosis, Parkinson’s disease,osteoporosis, osteoarthritis, osteopenia, and metabolic syndromes(including, but not limited to diabetes, obesity, nutritional disorders,organ atrophy, chronic obstructive pulmonary disease, and anorexia).Additional diseases, disorders, and conditions that can be prevented,treated and/or ameliorated using compositions of the present inventioninclude sepsis, chronic heart failure, benign and malignantpheochromocytoma, uterine fibroids/leiomyomata, preeclampsia, keloidsand hypertrophic scars, and pulmonary artery hypertension.

Improved Specificity of Binding and Activity

The present invention includes methods for increasing musclestrength/power and/or muscle mass and/or muscle function in a subject,or for treating a disease or disorder characterized by decreased musclemass or strength in a subject, or for treating a disease or disordercaused by, promoted by, or aggravated by Activin A activity, withoutcausing adverse effects associated with the administration of moleculeswhich bind multiple (e.g., 3 or more) ActRIIB ligands. In other words,methods using anti-Activin A antibodies or antigen binding proteinsthereof (e.g., wherein the anti-Activin A antibody only significantlybinds to Activin A) may treat a disease or disorder without causingunwanted or adverse effects seen with molecules which bind multipleActRIIB ligands. For example, the clinical molecule referred to asACE-031 (Acceleron Pharma, Inc., Cambridge, MA) is a multimer consistingof the extracellular portion of ActRIIB fused to an IgG Fc domain (thismolecule is also referred to herein as “ActRIIB-Fc”). ActRIIB-Fc bindsActivin A as well as other ActRIIB ligands such as, e.g., Activin B,GDF8, GDF11, BMP9, BMP10, and TGFβ, and is known to cause variousadverse effects when administered to human patients. Significantly, thepresent inventors have unexpectedly discovered that specificallyinhibiting Activin A and GDF8 (e.g., by administering an anti-Activin Aantibody and an anti-GDF8 antibody), while not inhibiting other ActRIIBligands such as Activin B, GDF11, BMP9, BMP10, and TGFβ, results in anincrease in muscle mass that is at least equivalent to that observed byadministration of ActRIIB-Fc, without causing the adverse effectsassociated with binding agents such as ActRIIB-Fc.

Combination Therapies and Formulations

The present invention includes compositions and therapeutic formulationscomprising any of the anti-Activin A antibodies described herein incombination with one or more additional therapeutically activecomponents, and methods of treatment comprising administering suchcombinations to subjects in need thereof. The present invention alsoincludes compositions and therapeutic formulations comprising any of theanti-Activin A antibodies described herein in combination with one ormore additional therapeutically active components, and methods oftreatment comprising administering such combinations to subjects in needthereof. For example, the anti-Activin A antibodies of the invention mayalso be administered and/or co-formulated in combination withantivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen,antioxidants, metal chelators, IFN-gamma, and/or NSAIDs. Theanti-Activin A antibodies of the invention may also be administered aspart of a treatment regimen that also includes radiation treatmentand/or conventional chemotherapy (e.g., in the context of methods oftreating cancer or inhibiting tumor growth). Any of the aforementionedadditional therapeutically active components may be administered incombination with any of the anti-Activin A antibodies of the presentinvention for the treatment of any disease or disorder in whichadministration of an anti-Activin A antibody is beneficial, including,e.g., sarcopenia, cachexia, muscle injury, muscle wasting and muscleatrophy. Any of the aforementioned additional therapeutically activecomponents may also be administered in combination with any of theanti-Activin A antibodies of the present invention along with a GDF8inhibitor (e.g., an anti-GDF8 antibody).

The additional therapeutically active component(s) may be administeredto a subject prior to administration of an anti-Activin A antibody ofthe present invention. For example, a first component may be deemed tobe administered “prior to” a second component if the first component isadministered 1 week before, 72 hours before, 60 hours before, 48 hoursbefore, 36 hours before, 24 hours before, 12 hours before, 6 hoursbefore, 5 hours before, 4 hours before, 3 hours before, 2 hours before,1 hour before, 30 minutes before, 15 minutes before, 10 minutes before,5 minutes before, or less than 1 minute before administration of thesecond component. In other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject afteradministration of an anti-Activin A antibody of the present invention.For example, a first component may be deemed to be administered “after”a second component if the first component is administered 1 minuteafter, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutesafter, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5hours after, 6 hours after, 12 hours after, 24 hours after, 36 hoursafter, 48 hours after, 60 hours after, 72 hours after administration ofthe second component. In yet other embodiments, the additionaltherapeutically active component(s) may be administered to a subjectconcurrent with administration of anti-Activin A antibody of the presentinvention. “Concurrent” administration, for purposes of the presentinvention, includes, e.g., administration of an anti-Activin A antibodyand an additional therapeutically active component to a subject in asingle dosage form, or in separate dosage forms administered to thesubject within about 30 minutes or less of each other. If administeredin separate dosage forms, each dosage form may be administered via thesame route (e.g., both the anti-Activin A antibody and the additionaltherapeutically active component may be administered intravenously,subcutaneously, intravitreally, etc.); alternatively, each dosage formmay be administered via a different route (e.g., the anti-Activin Aantibody may be administered locally (e.g., intravitreally) and theadditional therapeutically active component may be administeredsystemically). In any event, administering the components in a singledosage from, in separate dosage forms by the same route, or in separatedosage forms by different routes are all considered “concurrentadministration,” for purposes of the present disclosure. For purposes ofthe present disclosure, administration of an anti-Activin A antibody“prior to”, “concurrent with,” or “after” (as those terms are definedherein above) administration of an additional therapeutically activecomponent is considered administration of an anti-Activin A antibody “incombination with” an additional therapeutically active component).

The present invention includes pharmaceutical compositions in which ananti-Activin A antibody of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Dosage

The amount of active ingredient (e.g., anti-Activin A antibodies,anti-GDF8 antibodies given in combination with anti-Activin Aantibodies, or bispecific antibodies that specifically bind Activin Aand GDF8) that can be administered to a subject is, generally, atherapeutically effective amount. As used herein, the phrase“therapeutically effective amount” means a dose of antigen-specificbinding proteins and/or antigen-binding molecules that results in adetectable increase in one or more of the following parameters: bodyweight, muscle mass (e.g., tibialis anterior [TA] muscle mass,gastrocnemius [GA] muscle mass, quadriceps [Quad] muscle mass, etc.),muscle strength/power, and/or muscle function. For example, a“therapeutically effective amount” of an Activin A-specific bindingprotein and/or a GDF8-specific binding protein includes, e.g., an amountof Activin A-specific binding protein and/or GDF8-specific bindingprotein that, when administered to a test subject, causes an increase inTA or GA muscle mass of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60% or more, compared to control treated subjects, e.g., asillustrated in Example 7, herein.

In the case of antibodies of the present invention (e.g., anti-Activin Aantibodies, anti-GDF8 antibodies given in combination with anti-ActivinA antibodies, or bispecific antibodies that specifically bind Activin Aand GDF8), a therapeutically effective amount can be from about 0.05 mgto about 600 mg; e.g., about 0.05 mg, about 0.1 mg, about 1.0 mg, about1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg,about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg,about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg,about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg,about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg,about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg,about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg,about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg,about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg,about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg,about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg,about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg,about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg,about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg,about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg,about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg,about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg,about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg,about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, orabout 1000 mg, of the respective antibody.

The amount of antibody of the present invention (e.g., anti-Activin Aantibodies, anti-GDF8 antibodies given in combination with anti-ActivinA antibodies, or bispecific antibodies that specifically bind Activin Aand GDF8) contained within the individual doses may be expressed interms of milligrams of antibody per kilogram of patient body weight(i.e., mg/kg). For example, the anti-Activin A, anti-GDF8 and/oranti-Activin A/anti-GDF8 bispecific antibodies of the present inventionmay be administered to a patient at a dose of about 0.0001 to about 50mg/kg of patient body weight (e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg,5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 11.0mg/kg, 11.5 mg/kg, 12.0 mg/kg, 12.5 mg/kg, 13.0 mg/kg, 13.5 mg/kg, 14.0mg/kg, 14.5 mg/kg, 15.0 mg/kg, 15.5 mg/kg, 16.0 mg/kg, 16.5 mg/kg, 17.0mg/kg, 17.5 mg/kg, 18.0 mg/kg, 18.5 mg/kg, 19.0 mg/kg, 19.5 mg/kg, 20.0mg/kg, etc.).

The compositions of the present invention may comprise equal amounts ofActivin A-specific binding protein and GDF8-specific binding protein.Alternatively, the amount of Activin A-specific binding protein in thecomposition may be less than or greater than the amount of GDF8-specificbinding protein. A person of ordinary skill in the art, using routineexperimentation, will be able to determine the appropriate amounts ofthe individual components in the compositions of the present inventionnecessary to produce a desired therapeutic effect.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an active ingredient (e.g., an anti-Activin A antibody, ananti-GDF8 antibody administered in combination with an anti-Activin Aantibody, a pharmaceutical composition comprising a combination ofanti-Activin A antibody and any of the additional therapeutically activeagents mentioned herein, including, e.g., an anti-GDF8 antibody, or abispecific antibody that specifically bind Activin A and GDF8) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an active ingredient of theinvention. As used herein, “sequentially administering” means that eachdose of an active ingredient is administered to the subject at adifferent point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentinvention includes methods which comprise sequentially administering tothe patient a single initial dose of an active ingredient, followed byone or more secondary doses of the active ingredient, and optionallyfollowed by one or more tertiary doses of the active ingredient.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the active ingredient,e.g., anti-Activin A antibody of the invention or of a combinationtherapy of the invention, e.g., an anti-Activin A antibody and ananti-GDF8 antibody. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of theactive ingredient, e.g., anti-Activin A antibody, but generally maydiffer from one another in terms of frequency of administration. Incertain embodiments, however, the amount of the active ingredient, e.g.,anti- Activin A antibody, contained in the initial, secondary and/ortertiary doses varies from one another (e.g., adjusted up or down asappropriate) during the course of treatment. In certain embodiments, twoor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of the active ingredient, e.g., an anti-Activin A antibody, which is administered to a patient prior to theadministration of the very next dose in the sequence with no interveningdoses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an active ingredient of the invention, e.g., an anti- Activin Aantibody. For example, in certain embodiments, only a single secondarydose is administered to the patient. In other embodiments, two or more(e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered tothe patient. Likewise, in certain embodiments, only a single tertiarydose is administered to the patient. In other embodiments, two or more(e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered tothe patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens in which 2 to 6loading doses are administered to a patient a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the invention, if the loading doses areadministered at a frequency of once a month, then the maintenance dosesmay be administered to the patient once every six weeks, once every twomonths, once every three months, etc.).

Diagnostic Uses of the Antibodies

The anti-Activin A antibodies of the present invention may also be usedto detect and/or measure Activin A, or Activin A-expressing cells in asample, e.g., for diagnostic purposes. For example, an anti-Activin Aantibody, or fragment thereof, may be used to diagnose a condition ordisease characterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of Activin A. Exemplarydiagnostic assays for Activin A may comprise, e.g., contacting a sample,obtained from a patient, with an anti-Activin A antibody of theinvention, wherein the anti-Activin A antibody is labeled with adetectable label or reporter molecule. Alternatively, an unlabeledanti-Activin A antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure Activin A in asample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in Activin A diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of Activin A protein, orfragments thereof, under normal or pathological conditions. Generally,levels of Activin A in a particular sample obtained from a healthypatient (e.g., a patient not afflicted with a disease or conditionassociated with abnormal Activin A levels or activity) will be measuredto initially establish a baseline, or standard, level of Activin A. Thisbaseline level of Activin A can then be compared against the levels ofActivin A measured in samples obtained from individuals suspected ofhaving an Activin A-related disease or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Human Antibodies to Activin A

An immunogen comprising the Activin A protein (inhibin-βA dimer) wasadministered directly, with an adjuvant to stimulate the immuneresponse, to a VELOCIMMUNE® mouse comprising DNA encoding humanImmunoglobulin heavy and kappa light chain variable regions. Theantibody immune response was monitored by a Activin A-specificimmunoassay. When a desired immune response was achieved splenocyteswere harvested and fused with mouse myeloma cells to preserve theirviability and form hybridoma cell lines. The hybridoma cell lines werescreened and selected to identify cell lines that produce ActivinA-specific antibodies. Using this technique several anti-Activin Achimeric antibodies (i.e., antibodies possessing human variable domainsand mouse constant domains) were obtained. An exemplary antibodyobtained in this manner is H2aM10965N. The human variable domains fromthe chimeric antibodies were subsequently cloned onto human constantdomains to make fully human anti-Activin A antibodies as describedherein.

Anti-Activin A antibodies were also isolated directly fromantigen-positive B cells without fusion to myeloma cells, as describedin US 2007/0280945A1. Using this method, several fully humananti-Activin A antibodies (i.e., antibodies possessing human variabledomains and human constant domains) were obtained; exemplary antibodiesgenerated in this manner were designated as follows: H4H10423P,H4H10429P, H4H10430P, H4H10432P2, H4H10440P2, H4H10442P2, H4H10436P2,and H4H10446P2.

Certain biological properties of the exemplary anti-Activin A antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected anti-Activin A antibodies and theircorresponding antibody identifiers. The corresponding nucleic acidsequence identifiers are set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers SEQ ID NOs Antibody DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H4H10423P 2 4 6 8 10 12 1416 H4H10424P 18 20 22 24 26 28 30 32 H4H10426P 34 36 38 40 42 44 46 48H4H10429P 50 52 54 56 58 60 62 64 H4H10430P 66 68 70 72 74 76 78 80H4H10432P2 82 84 86 88 90 92 94 96 H4H10433P2 98 100 102 104 90 92 94 96H4H10436P2 106 108 110 112 90 92 94 96 H4H10437P2 114 116 118 120 90 9294 96 H4H10438P2 122 124 126 128 90 92 94 96 H4H10440P2 130 132 134 13690 92 94 96 H4H10442P2 138 140 142 144 146 148 150 152 H4H10445P2 154156 158 160 146 148 150 152 H4H10446P2 162 164 166 168 146 148 150 152H4H10447P2 170 172 174 176 146 148 150 152 H4H10448P2 178 180 182 184146 148 150 152 H4H10452P2 186 188 190 192 146 148 150 152 H4H10468P2194 196 198 200 146 148 150 152 H2aM10965N 202 204 206 208 210 212 214216

TABLE 2 Nucleic Acid Sequence Identifiers SEQ ID NOs AntibodyDesignation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H4H10423P 1 35 7 9 11 13 15 H4H10424P 17 19 21 23 25 27 29 31 H4H10426P 33 35 37 3941 43 45 47 H4H10429P 49 51 53 55 57 59 61 63 H4H10430P 65 67 69 71 7375 77 79 H4H10432P2 81 83 85 87 89 91 93 95 H4H10433P2 97 99 101 103 8991 93 95 H4H10436P2 105 107 109 111 89 91 93 95 H4H10437P2 113 115 117119 89 91 93 95 H4H10438P2 121 123 125 127 89 91 93 95 H4H10440P2 129131 133 135 89 91 93 95 H4H10442P2 137 139 141 143 145 147 149 151H4H10445P2 153 155 157 159 145 147 149 151 H4H10446P2 161 163 165 167145 147 149 151 H4H10447P2 169 171 173 175 145 147 149 151 H4H10448P2177 179 181 183 145 147 149 151 H4H10452P2 185 187 189 191 145 147 149151 H4H10468P2 193 195 197 199 145 147 149 151 H2aM10965N 201 203 205207 209 211 213 215

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M,” “H2aM,” “H4H”), followed by anumerical identifier (e.g. “10423,” “10424,” or “10426” as shown inTables 1 and 2), followed by a “P,” “P2” or “N” suffix. Thus, accordingto this nomenclature, an antibody may be referred to herein as, e.g., “H4H10423P,” “ H4H10432P2,” “H2aM10965N,” etc. The H1M, H2M and H4Hprefixes on the antibody designations used herein indicate theparticular Fc region isotype of the antibody. For example, an “H2aM”antibody has a mouse IgG2a Fc, whereas an “H4H” antibody has a humanIgG4 Fc. As will be appreciated by a person of ordinary skill in theart, an antibody having a particular Fc isotype can be converted to anantibody with a different Fc isotype (e.g., an antibody with a mouseIgG2a Fc can be converted to an antibody with a human IgG4, etc.), butin any event, the variable domains (including the CDRs) -which areindicated by the numerical identifiers shown in Table 1 - will remainthe same, and the binding properties are expected to be identical orsubstantially similar regardless of the nature of the Fc domain.

Control Constructs Used in the Following Examples

Anti-Activin A control molecules were included in the following Examplesfor comparative purposes. The control antibody designated herein asControl 1 is a human anti-Activin A antibody with heavy and light chainvariable domain sequences of “A1” as set forth in US 8,309,082. Control2 is an anti-human Activin Receptor Type II B antibody (anti-ActR2B mAb)disclosed as MOR8159 in U.S. Pat. Application No. 2012/0237521 A1.Control 3 is a murine anti-Activin A monoclonal antibody from R&DSystems, Minneapolis, MN (catalog number MAB3381). Control 4 is anActivin Type IIB receptor-Fc fusion molecule (a soluble Activin RIIBreceptor extracelullar domain produced with a C-terminal human IgG1 Fcfusion protein (E23-P133 of NP_001097 followed by a Gly-Ser linkerfollowed by a C-terminal human IgG1 Fc fusion), the sequence of which isprovided as SEQ ID NO:227.

Example 3. Antibody Binding to Human Activin A as Determined by SurfacePlasmon Resonance

Binding affinities and kinetic constants for antigen binding to selectedpurified anti-human Activin A monoclonal antibodies were determinedusing a real-time surface plasmon resonance biosensor (BIACORE™ T200 orBIACORE™ 4000, GE Healthcare Life Sciences, Piscataway, NJ) assay at 25°C. and 37° C. Antibodies, expressed as either mouse Fc (prefix H2aM) orhuman Fc (prefix H4H), were captured on their respective anti-Fc sensorsurfaces (mAb capture format). Anti-Activin A antibodies were capturedon either a goat anti-mouse IgG polyclonal antibody (GE Healthcare,#BR-1008-38) or a mouse anti-human IgG monoclonal antibody (GEHealthcare, #BR-1008-39) surface created through direct amine couplingto a BIACORE™ CM5 sensor chip. Kinetic experiments were carried outusing either HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Surfactant P20, at pH 7.4) or PBS-P (10 mM Sodium Phosphate, 2.7 mM KCI,137 mM NaCl, 0.02% NaN3, 0.05% Surfactant P20, pH 7.4), as both therunning buffer and the sample buffer. Antigen-antibody association rateswere measured by injecting various concentrations (4-fold dilutionsranging from 50 to 0.2 nM) of either Activin A (R&D Systems, #338-AC-050/CF), Activin B (R&D Systems, # 659-AB-025/CF), Activin AB(R&D Systems, # 1006-AB-005), Activin AC (R&D Systems, # 4879-AC/CF), orInhibin E (Novus Biologicals, #H00083729-P01) over the captured antibodysurface. Antibody-antigen association was monitored for 240 secondswhile dissociation in buffer was monitored for 600 seconds. Kineticassociation and dissociation rate constants were determined byprocessing and fitting the data using Scrubber software version 2.0c.Binding equilibrium dissociation constants (K_(D)) and dissociativehalf-lives (t_(½)) were then calculated from the kinetic rate constantsas: K_(D) (M) = k_(d) / k_(a) and t_(½) (min) = [In2/(60*k_(d))].Kinetic binding parameters for different anti-Activin A monoclonalantibodies are shown in Tables 3 to 10. (NB = no binding observed underthe conditions used; NT = not tested).

TABLE 3 Binding Characteristics of Anti-Activin A Antibodies to ActivinA at 25° C. Antibody Amount of mAb Captured (RU ± SE) Activin A-20 nM(RU) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P 86.2± 0.7 19.4 3.33E+06 1.09E-04 3.26E-11 106.4 H4H10424P 337 82 3.14E+067.19E-04 2.29E-10 16 H4H10426P 81 23 1.18E+07 7.00E-04 5.95E-11 16H4H10429P 115.2 ± 1 24.9 7.82E+06 6.39E-05 8.17E-12 180.8 H4H10430P 90.3± 4.2 19.4 4.75E+07 1.67E-04 3.52E-12 69 H4H10432P2 109.6 ± 1.2 20.71.57E+07 5.00E-05 ≤ 3.18E-12 ≥231 H4H10433P2 102 16 1.42E+07 5.77E-044.06E-11 20 H4H10436P2 113.6 ± 0.6 23.2 8.85E+06 1.68E-04 1.90E-11 68.7H4H10437P2 167 30 1.58E+07 2.13E-03 1.34E-10 5 H4H10438P2 124 251.20E+07 5.88E-04 4.92E-11 20 H4H10440P2 79.2 ± 0.7 12.9 3.76E+069.28E-05 2.47E-11 124.5 H4H10442P2 139.3 ± 1 31.3 1.10E+07 5.00E-05 ≤4.55E-12 ≥231 H4H10445P2 149 43 2.40E+06 5.00E-05 ≤ 2.08E-11 ≥231H4H10446P2 104.6 ± 0.7 24.1 1.29E+07 5.00E-05 ≤ 3.88E-12 ≥231 H4H10447P2164 43 2.36E+06 5.00E-05 ≤ 2.12E-11 ≥231 H4H10448P2 244 64 4.76E+065.00E-05 ≤ 1.05E-11 ≥231 H4H10452P2 191 55 4.69E+06 5.00E-05 ≤ 1.07E-11≥231 H4H10468P2 93 ± 0.1 21.7 7.86E+06 5.00E-05 ≤ 6.36E-12 ≥231H2aM10965N 393 76 1.48E+06 1.10E-03 7.45E-10 10 Control 1 84.7 ± 0.315.9 7.26E+06 9.92E-05 1.37E-11 116.4 For k_(d) values that areitalicized, no dissociation of the analyte was observed under theseexperimental conditions, and the value of k_(d) was therefore fixed at5.0E-05 s⁻¹

TABLE 4 Binding Characteristics of Anti-Activin A Antibodies to toActivin A at 37° C. Antibody Amount of mAb Captured (RU ± SE) ActivinA-20 nM (RU) k_(a) (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P101 ± 1.4 25.2 3.95E+06 5.00E-05 ≤1.26E-11 ≥231 H4H10424P 231 584.59E+06 3.64E-03 7.94E-10 3 H4H10426P 71 21 1.61E+07 1.98E-03 1.23E-106 H4H10429P 150.8 ± 5.3 31.4 1.33E+07 5.00E-05 ≤3.75E-12 ≥231 H4H10430P109.3 ± 1.3 25.0 3.80E+07 1.51E-04 3.97E-12 76.5 H4H10432P2 141.8 ± 1.625.1 2.30E+07 5.00E-05 ≤2.18E-12 ≥231 H4H10433P2 85 12 2.00E+07 1.07E-035.37E-11 11 H4H10436P2 139.8 ± 1.4 29.4 1.49E+07 5.00E-05 ≤3.35E-12 ≥231H4H10437P2 115 20 2.04E+07 4.68E-03 2.29E-10 2 H4H10438P2 99 18 1.87E+072.38E-03 1.27E-10 5 H4H10440P2 98.6 ± 1.1 15.3 6.37E+06 3.28E-045.15E-11 35.2 H4H10442P2 181 ± 2.5 40.5 1.44E+07 5.00E-05 ≤3.48E-12 ≥231H4H10445P2 120 36 4.33E+06 5.00E-05 ≤ 1.15E-11 ≥231 H4H10446P2 137.2 ±1.7 31.5 1.54E+07 5.00E-05 ≤ 3.25E-12 ≥231 H4H10447P2 126 36 4.69E+065.00E-05 ≤ 1.07E-11 ≥231 H4H10448P2 175 49 7.86E+06 5.00E-05 ≤ 6.36E-12≥231 H4H10452P2 146 43 7.94E+06 5.00E-05 ≤ 6.30E-12 ≥231 H4H10468P2 98.7± 0.7 24.5 1.22E+07 5.00E-05 ≤ 4.10E-12 ≥231 H2aM10965N 435 80 2.35E+064.15E-03 1.77E-09 3 Control 1 93.9 ± 0.7 18.0 8.99E+06 5.00E-05 ≤5.56E-12 ≥231 For k_(d) values that are italicized, no dissociation ofthe analyte was observed under these experimental conditions, and thevalue of k_(d) was therefore fixed at 5.0E-05 s⁻¹

TABLE 5 Binding Characteristics of Anti-Activin A Antibodies to ActivinB at 25° C. Antibody Amount of mAb Captured (RU ± SE) 50 nM Ag Bound(RU) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P 83.1± 0.6 4.7 4.89E+05 3.02E-02 6.18E-08 0.4 H4H10429P 112.3 ± 0.7 26.43.49E+06 1.31E-02 3.75E-09 0.9 H4H10432P2 104.4 ± 1.8 5.1 NB NB NB NBH4H10436P2 110.8 ± 3.9 32.8 9.52E+06 5.28E-04 5.54E-11 21.9 H4H10440P275.7 ± 0.8 18.8 1.06E+06 1.16E-03 1.09E-09 10.0 H4H10442P2 136 ± 0.7 3.4NB NB NB NB H4H10430P 88 ± 0.5 3.9 NB NB NB NB H4H10446P2 101.5 ± 0.43.6 NB NB NB NB H4H10468P2 92.5 ± 0.2 6.2 NB NB NB NB Control 1 84.1 ±0.3 6.4 NB NB NB NB

TABLE 6 Binding Characteristics of Anti-Activin A Antibodies to ActivinB at 37° C. Antibody Amount of mAb Captured (RU ± SE) 50 nM Ag Bound(RU) k_(a) (M⁻¹s⁻¹) k_(a) (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P 96 ±1.2 4.4 NB NB NB NB H4H10429P 142.8 ± 1.3 25.3 3.43E+06 3.43E-029.98E-09 0.3 H4H10432P2 134.1 ± 1.7 5.1 NB NB NB NB H4H10436P2 132 ± 1.438.1 9.78E+06 1.36E-03 1.39E-10 8.5 H4H10440P2 94 ± 4.5 20.9 1.28E+064.19E-03 3.29E-09 2.8 H4H10442P2 173.1 ± 1.4 4.4 NB NB NB NB H4H10430P105.8 ± 1.3 3.6 NB NB NB NB H4H10446P2 131.4 ± 1.2 3.8 NB NB NB NBH4H10468P2 95.5 ± 1 3.4 NB NB NB NB Control 1 90.2 ± 0.9 2.7 NB NB NB NB

TABLE 7 Binding Characteristics of Anti-Activin A Antibodies to ActivinAB at 25° C. Antibody Amount of mAb Captured (RU ± SE) 50 nM Ag Bound(RU) k_(a) (M⁻¹s⁻¹) k_(a) (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P 81.3± 0.5 14.7 6.13E+05 2.03E-02 3.31E-08 0.6 H4H10429P 110.7 ± 0.5 40.04.53E+06 1.03E-04 2.28E-11 111.7 H4H10432P2 101.2 ± 1.6 38.3 4.00E+062.27E-03 5.68E-10 5.1 H4H10436P2 107.5 ± 0.3 28.2 7.66E+06 2.61E-043.41E-11 44.2 H4H10440P2 73.7 ± 0.4 15.5 2.97E+06 5.26E-04 1.77E-10 22.0H4H10442P2 133.3 ± 0.6 34.6 5.53E+06 1.77E-03 3.20E-10 6.5 H4H10430P86.9 ± 0.5 33.0 1.17E+07 2.17E-04 1.85E-11 53.3 H4H10446P2 99.8 ± 0.431.9 4.99E+06 4.06E-03 8.15E-10 2.8 H4H10468P2 92.1 ± 0.2 34.7 3.76E+062.09E-03 5.56E-10 5.5 Control 1 83.5 ± 0.6 31.1 3.44E+06 2.83E-048.22E-11 40.9

TABLE 8 Binding Characteristics of Anti-Activin A Antibodies to ActivinAB at 37° C. Antibody Amount of mAb Captured (RU ± SE) 50 nM Ag Bound(RU) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P 90.8± 1.2 21.7 8.80E+05 2.13E-02 2.42E-08 0.5 H4H10429P 137.7 ± 1.2 50.06.47E+06 4.88E-04 7.55E-11 23.6 H4H10432P2 127.7 ± 1.3 44.4 5.40E+065.92E-03 1.10E-09 2.0 H4H10436P2 126.8 ± 0.8 33.9 1.03E+07 4.58E-044.43E-11 25.2 H4H10440P2 88.9 ± 1.7 17.7 5.20E+06 1.63E-03 3.14E-10 7.1H4H10442P2 166.5 ± 1.7 45.9 9.17E+06 4.25E-03 4.64E-10 2.7 H4H10430P101.6 ± 1.2 41.0 1.01E+07 5.41E-04 5.35E-11 21.3 H4H10446P2 126.6 ± 1.241.5 6.08E+06 8.17E-03 1.34E-09 1.4 H4H10468P2 92.2 ± 0.8 34.5 5.03E+064.43E-03 8.80E-10 2.6 Control 1 86.4 ± 0.6 29.3 3.77E+06 7.38E-041.96E-10 15.7

TABLE 9 Binding Characteristics of Anti-Activin A Antibodies to ActivinAC at 25° C. Antibody Amount of mAb Captured (RU ± SE) 50 nM Ag Bound(RU) k_(a) (M⁻¹s⁻¹) k_(a) (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P 79.9± 0.4 -0.8 NB NB NB NB H4H10429P 108.9 ± 0.5 28.0 9.13E+05 9.10E-059.97E-11 126.9 H4H10432P2 101.6 ± 0.7 34.9 6.29E+05 1.87E-03 2.98E-096.2 H4H10436P2 106.7 ± 0.4 30.1 6.98E+05 1.56E-03 2.24E-09 7.4H4H10440P2 73.5 ± 0.4 11.8 5.13E+05 2.27E-04 4.42E-10 50.8 H4H10442P2132.5 ± 3.1 18.6 1.31E+06 2.05E-03 1.57E-09 5.6 H4H10430P 85.1 ± 0.323.6 1.23E+06 1.09E-02 8.86E-09 1.1 H4H10446P2 96.9 ± 0.5 12.6 1.04E+061.22E-02 1.18E-08 0.9 H4H10468P2 91.4 ± 0.3 17.2 7.98E+05 5.92E-037.41E-09 2.0 Control 1 82.5 ± 0.3 22.3 5.58E+05 2.25E-03 4.03E-09 5.1

TABLE 10 Binding Characteristics of Anti-Activin A Antibodies to ActivinAC at 37° C. Antibody Amount of mAb Captured (RU ± SE) 50 nM Ag Bound(RU) k_(a) (M⁻¹s⁻¹) k_(a) (s⁻¹) K_(D) (Molar) t_(½) (min) H4H10423P 85.9± 1.1 0.0 NB NB NB NB H4H10429P 132.6 ± 1.2 35.7 1.34E+06 6.20E-044.62E-10 18.6 H4H10432P2 123.8 ± 1.4 34.6 7.22E+05 9.02E-03 1.25E-08 1.3H4H10436P2 122.9 ± 1.3 32.6 8.81E+05 3.31E-03 3.75E-09 3.5 H4H10440P286.6 ± 2.7 13.3 7.18E+05 7.55E-04 1.05E-09 15.3 H4H10442P2 160.1 ± 1.521.4 1.46E+06 5.99E-03 4.10E-09 1.9 H4H10430P 96.8 ± 1 25.3 1.20E+062.00E-02 1.67E-08 0.6 H4H10446P2 120.3 ± 1 14.4 9.59E+05 2.16E-022.25E-08 0.5 H4H10468P2 88.4 ± 0.8 10.7 7.19E+05 1.24E-02 1.73E-08 0.9Control 1 83.2 ± 0.9 15.6 6.51E+05 6.52E-03 1.00E-08 1.8

As show n in Tables 3 and 4, anti-Activin A antibodies of the inventionbound to Activin A with K_(D) values ranging from less than 3.18 pM(i.e., ≤ 3.18E-12) to 745 pM (i.e., 7.45E-10) at 25° C. and with K_(D)values ranging from less than 2.18 pM (i.e., ≤ 2.18E-12) to 1.77 nM(1.77E-09) at 37° C. As shown in Tables 5 and 6, several of theanti-Activin A antibodies (i.e., H4H10432P2, H4H10442P2, H4H10430P2,H4H10446P2, and H4H10468P2) demonstrated no measurable binding toActivin B at 25° C. or 37° C. Some of the antibodies demonstratedmeasurable binding to Activin AB with K_(D) values ranging fromapproximately 18.5 pM (i.e., 1.85E-11) to 33.1 nM (i.e., 3.31E-08) at25° C. (Table 7) and from approximately 44.3 pM (i.e., 4.43E-11) to 24.2nM (i.e., 2.42E-08) at 37° C. (Table 8). Some of the antibodiesdemonstrated measurable binding to Activin AC with K_(D) values rangingfrom approximately 99.7 pM (i.e., 9.97E-11) to 11.8 nM (i.e., 1.18E-08)at 25° C. (Table 9) and from approximately 462 pM (i.e., 4.62E-10) to22.5 nM (i.e., 2.25E-08) at 37° C. (Table 10). Furthermore, none of thetested anti-Activin A antibodies of the invention demonstratedmeasurable binding to Inhibin E (data not shown).

Example 4. Antibody Binding to TGF-beta Family Members as Determined bySurface Plasmon Resonance

Activin A mAbs were tested for binding cross-reactivity to a panel ofTGF-beta family members. For the binding experiment, a BIACORE™ 4000real-time surface plasmon resonance biosensor instrument was used. Theantibodies H4H10429P, H4H10430P, H4H10436P2, H4H10442P2, H4H10446P2;Control 4 (the ActR2B soluble ecto domain protein produced with aC-terminal human IgG1 Fc tag (ActR2B-hFc; SEQ ID NO:227)); and anisotype control antibody were captured on a BIACORE™ CM4 biosensor chipthat was first derivatized by amine coupling with a monoclonal mouseanti-human Fc antibody (GE, Catalog# BR-1008-39). All BIACORE™ bindingstudies were performed in HBS-T running buffer (0.01 M HEPES pH 7.4, 0.5M NaCl, 3 mM EDTA, 0.5 mg/ml bovine serum albumin, 0.05% v/v SurfactantP20). Human TGF-beta family member ligands were purchased from R&Dsystems (Activin A, #338-AC; Activin B, #659-AB; Activin AB, #1066-AB;Activin AC, #4879-AC; BPM2, #355-BM; hBMP4, #314-BP; hBMP6, #507-BP;hBMP7, #354-BP; hBMP9, #3209-BP; hBMP10, #2926-BP; hGDF8, #788-G8;hGDF11, #1958-GD). All binding measurements were performed at 37° C.Capture levels ranging from 60 - 200 resonance units (RUs) were obtainedfor each of the antibodies or the soluble receptor. Over the capturedantibody surface was injected the TGF-beta family ligands throughconcentrations ranging from 3.1 nM to 200 nM. Binding values for the 200nM analyte injections are shown in Table 11.

TABLE 11 Binding of anti-Activin A monoclonal antibodies to human TGF-βfamily ligands at 37° C. Binding response (resonance units) for 200 nMof TGF-beta family ligand injected over captured antibody sensor surfaceTGF-beta family ligand tested H4H10429P H4H10430P H4H10436P2 H4H10442P2H4H10446P2 H4H8925C Control 4 (ActR2B-hFc) (Positive Control) IsotypeControl mAb Activin A 56.8 69.1 56.5 63.9 53.2 67.6 70.9 -0.1 Activin B51.5 0.4 60.1 -1.8 2.6 0.3 68.0 1.9 Activin AB 76.5 95.0 54.3 65.2 66.7102.0 59.5 -0.1 Activin AC 43.1 34.8 55.6 14.2 15.2 32.5 59.4 -0.1 hBMP23.6 -1.7 14.9 -2.3 3.3 -4.0 36.9 -1.0 hBMP4 1.1 -0.6 19.3 -0.7 0.8 -0.526.4 0.4 hBMP6 4.6 5.7 4.0 1.1 5.3 4.8 86.3 5.1 hBMP7 9.2 6.4 13.6 1.55.7 4.5 64.2 4.3 hBMP9 33.4 -0.6 11.7 0.0 -0.3 -0.1 32.3 -1.0 hBMP1032.4 0.3 22.5 -0.7 0.5 0.0 34.2 0.3 GDF8 -0.4 -0.1 -0.5 0.5 0.7 -0.125.8 0.5 GDF11 1.6 3.0 0.0 1.0 1.8 3.3 24.2 3.0

The observed binding responses of the captured activin A antibodies tothe injected TGF-beta family ligands at 200 nM could be compared to thebinding responses of a negative control antibody (Isotype Control mAb),which provides a measure of background-level non-specific binding, andto the binding responses of ActR2B-hFc, which was observed to bind tothe entire panel of TGF-beta family members tested and therefore servesas a positive control ligand-binding protein (Table 11). From thiscomparison, it was found that several of the antibodies (e.g., H4H10430,H4H10442, H4H20446) bound to Activin A, Activin AB, Activin AC but notappreciably to Activin B or to the BMP or GDF ligands. It was also foundthat some of the antibodies bound with broader cross-reactivity toadditional TGF-beta family ligands. For example, H4H10429P boundappreciably to Activin A, Activin B, Activin AB, Activin AC and also toBMP9 and BMP10. H4H10436P2 showed appreciable binding to Activin A,Activin B, Activin AB, Activin AC, BMP2, BMP4, BMP7, BMP9, and BMP10.From these data it is shown that antibodies with different bindingspecificities to TGF-beta family ligands can be obtained afterimmunizing mice with the Activin A ligand.

Example 5. Cross-Competition Analysis of anti-Activin A Antibodies

A cross-competition assay was conducted to assess the ability of a panelof 9 antibodies (H4H10446P2, H4H10468P2, H4H10442P2, H4H10423P,H4H10430P, H4H10429P, H4H10432P2, H4H10436P2 and H4H10440P2) to competewith one another for binding to human Activin A. Two isotype controlantibodies and two control Activin A antibodies, Control 1 (a humananti-Activin A antibody with heavy and light chain variable domainsequences of “A1” as set forth in US 8,309,082) and Control 3 (MAB3381,available from R&D Systems, Inc., Minneapolis, MN) were also included inthe assays. All assays were performed at 25° C. with a microtiter plateshaking rate of 1000 rpm in OCTET® HBST buffer (0.01 M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, 0.1 mg/mL BSA) according tomanufacturer’s instructions (ForteBio Corp., Menlo Park, CA). Briefly,an amount of anti-Activin A antibody giving a binding resopnse ofapproximately 1.8 nm was captured onto anti-human Fc antibody coatedOCTET® sensor tips (Fortebio, # 18-0015) by dipping the tips for 5minutes in a 10 µg/mL solutions of each anti-Activin A antibody. Anyremaining anti-hFc binding sites on the tips were blocked by incubatingthe tips in a 50ug/mL solution of irrelevant antibody for 5 minutes.Sensor tips were then submerged into wells containing a solution of 50nM Activin A (R&D Systems, # 338-AC/CF) pre-bound with 1 µM of a secondanti-Activin A antibody. Binding of the second Activin A antibody /Activin A solution to the Activin A antibody coated sensor tip wasmonitored for 5 minutes at 1000 rpm. The response of the mAb/Activin Acomplex binding to the anti-Activin A coated sensor tip was compared andcompetitive/non-competitive behavior of different anti-Activin Amonoclonal antibodies was determined. Results are illustrated in FIG. 1.

In FIG. 1 , competitive binding responses are shown in black or lightgray shading and indicate that the corresponding antibody pairs competewith one another for binding to Activin A. Light gray boxes with blackfont represent binding response for self-competition between the sameantibodies. Black boxes with white font represent antibodies thatcompete for Activin A binding in both directions, independent of theorder of binding. Dark grey boxes with black font represent readings forisotype control (i.e., non-binding) antibodies, indicating a lack ofbinding of isotype control antibodies to anti-Activin A antibody-ActivinA complexes (when isotype control antibodies are bound to the OCTET®sensor tip) or the lack of binding of isotype control antibodies toActivin A (when isotype control antibodies are used as the secondantibody in wells with Activin A).. White boxes with black fontrepresent no competition between antibodies, which suggests theantibodies have distinct binding epitopes on Activin A.

Four antibodies, H4H10446P2, H4H10468P2, H4H10442P2, and H4H10423P,bi-directionally compete with each other for binding to Activin A.Additionally, these four antibodies do not compete with Control 1 orControl 3 for binding. Three of these four Activin A antibodies,H4H10446P2, H4H10468P2, and H4H10442P2, do not cross compete with anyother Activin A antibodies. One of the four antibodies (H4H10423P) alsobidirectionally competes with H4H10430P for binding to Activin A. Fiveantibodies, H4H10430P, H4H10429, H4H10432P2, H4H10436P2, and H4H10440P2,bi-directionally compete with each other for binding to Activin A, aswell as with Control 1 and Control 3. Four of these five antibodies(i.e., H4H10429, H4H10432P2, H4H10436P2, and H4H10440P2) do not crosscompete with any other Activin A antibodies, whereas H4H10423P alsocross-competes with H4H10430P, as noted above.

The results of this Example indicate that the anti-Activin A antibodiesof the invention can be grouped into two distinct “bins” based onepitope binding characteristics: Bin 1 includes H4H10423P, H4H10446P2,H4H10468P2 and H4H10442P2. Bin 2 includes H4H10429, H4H10430P,H4H10432P2, H4H10436P2, and H4H10440P2. Further, one antibody from eachbin, i.e., H4H10423P and H4H10430P, cross-compete with each other. Theresults of this Example suggest that the antibodies of Bin 1 bind todistinct regions on Activin A than the antibodies of Bin 2.

Example 6. Inhibition of Activin A-Mediated Receptor Activation and SMADComplex Signaling with Anti-Activin A Antibodies

To further characterize anti-Activin A antibodies of the presentinvention, a bioassay was developed to detect the activation of theactivin Type IIA and IIB receptors (ActRIIA and ActRIIB, respectively)and the subsequent phosphorylation and activation of an Activin Type Ireceptor. The interaction between ActRIIA and ActRllB and activin leadsto the induction of diverse cellular processes including growthregulation, metastatis of cancer cells and differentiation of embryonicstem cells (Tsuchida, K. et al., Cell Commun Signal 7:15 (2009)).Phosphorylation and activation of the Type I receptor leads tophosphorylation of SMAD 2 and 3 proteins which form activated SMADcomplexes leading to transcriptional regulation of genes.

To detect the activation of the SMAD complex signal transduction pathwayvia activin binding to activin Type II receptors, a human A204rhabdomyosarcoma cell line (ATCC, # HTB-82) was transfected with a Smad⅔-luciferase reporter plasmid (CAGAx12-Luc; Dennler, 1998) to create theA204/CAGAx12-Luc cell line. A204/CAGAx12-Luc cells were maintained inMcCoy’s 5A (Irvine Scientific, # 9090) supplemented with 10% fetalbovine serum (FBS), penicillin/streptomycin/glutamine and 250 µg/mL ofG418. For the bioassay, A204/CAGAx12-Luc cells were seeded onto 96-wellassay plates at 10,000 cells/well in low serum media, 0.5%FBS andOPTIMEM™ (Invitrogen, #31985-070), and incubated at 37° C. and 5% CO₂overnight. To determine the ligand dose response, Activin A (R&DSystems, #338-AC), Activin B (R&D Systems, #659-AB), Activin AB (R&DSystems, #1066-AB) and Activin AC (R&D Systems, #4879-AC/CF) wereserially diluted at 1:3 from 100 to 0.002 nM and added to cells startingalong with a control containing no Activin. Activin A, Activin B,Activin AB, and Activin AC were observed to activate theA204/CAGAx12-Luc cell line with EC₅₀ values of 99 pM, 47 pM, 19 pM, and4.4 nM, respectively. To measure inhibition, antibodies were seriallydiluted at 1:3 starting from 100 to 0.002 nM, 1000 to 0.02 nM, or 300 to0.005 nM including control samples containing either an appropriateisotype control antibody or no antibody and added to cells with aconstant concentration of 100 pM Activin A, 50 pM Activin B, 30 pMActivin AB or 4 nM Activin AC. Also used as a positive blocking controlin this assay was Control 4 (ActRIIB-hFc; SEQ ID No:227). After 5.5hours of incubation in 37° C. and 5% CO₂, ONEGLO™ substrate (Promega, #E6051) was added and then luciferase activity was detected using aVICTOR™ X (Perkin Elmer) instrument. The results were analyzed usingnonlinear regression (4-parameter logistics) with Prism 5 software(GraphPad).

As shown in Table 12, anti-Activin A antibodies of the invention blocked100 pM of Activin A with IC₅₀ values ranging from 39 pM to 3.5 nM, whileControl 1 blocked with an IC₅₀ value of 83 pM. A subset of theanti-Activin A antibodies of the invention were tested for blockingActivin B, AB, and AC. Four of the 9 antibodies tested blocked 50 pM ofActivin B with IC₅₀ values ranging from 130 pM to 100 nM. Fiveantibodies of the invention that were tested for Activin B blockade onlyblocked at high antibody concentrations, while Control 1 did not showany measurable Activin B blockade. Eight antibodies of the inventiontested blocked 30 pM of Activin AB with IC₅₀ values ranging from 100 pMto 8.2 nM, while Control 4 blocked with an IC₅₀ value of 540 pM. Oneantibody, H4H10423P, only demonstrated weak blockade of Activin AB.Seven of the 8 antibodies tested blocked 4 nM of Activin AC with IC₅₀values ranging from 580pM to 6.5 nM, while Control 4 blocked with anIC₅₀ value of 1.1 nM. One antibody, H4H10423P, did not demonstrate anyblockade of Activin AC. Both mouse IgG (mlgG isotype control) and humanIgG (hlgG isotype control) negative controls did not block ligandactivation of the receptors.

TABLE 12 Inhibition of Activin A, Activin B, Activin AB, and Activin ACby anti-Activin A antibodies (IC50 [M]) Constant: Activin A Activin BActivin AB Activin AC Antibody H4H10423P 2.0E-10 Weak BlockerNon-Blocker H4H10424P 7.6E-10 H4H10426P 2.3E-10 H4H10429P 1.6E-107.9E-08 2.9E-10 5.8E-10 H4H10430P 6.1E-11 Block at High Conc. 1.0E-109.3E-10 H4H10432P2 1.1E-10 Block at High Conc. 8.0E-10 2.8E-09H4H10433P2 1.5E-10 1.0E-07 H4H10436P2 2.0E-10 1.3E-10 1.4E-10 1.3E-09H4H10437P2 2.9E-10 Block at High Conc. H4H10438P2 2.6E-10 H4H10440P22.8E-10 5.2E-09 4.3E-10 7.5E-10 H4H10442P2 5.6E-11 2.2E-09 6.5E-09H4H10445P2 5.3E-11 H4H10446P2 4.7E-11 Block at High Conc. 8.2E-095.6E-09 H4H10447P2 7.8E-11 H4H10448P2 4.6E-11 H4H10452P2 5.8E-11H4H10468P2 3.9E-11 Block at High Conc. 2.3E-09 3.4E-09 H2aM10965N3.5E-09 mlgG isotype control Non-Blocker hlgG isotype controlNon-Blocker Non-Blocker Non-Blocker Non-Blocker Control 1 8.3E-11Non-Blocker 5.4E-10 1.1E-09

. The bioassay using A204/CAGAx12-Luc cells could also be stimulated byGDF8 (R&D Systems, Cat # 788-G8/CF) and GDF11 (R&D Systems, Cat #1958-GD-010/CF). To test for functional inhibition of these ligands withactivin A antibodies, the assay was performed using conditions describedabove but substituting GDF8 or GDF11 for the activating ligand, whichresulted in EC50 values of 188 pM and 84 pM, respectively. In thisassay, activation by a constant concentration of 0.50 nM GDF8 or 0.40 nMGDF11 was completely blocked by Control 4 with IC₅₀ values of 298 pM and214 pM, respectively. Using these same constant concentrations ofligands, no inhibition of either GDF8 or GDF11 was observed by theactivin A antibodies, H4H10446P2 and H4H10430P, when tested at up to 100nM of the antibodies. On a separate day, the activin A antibodiesH4H10429P and H4H10436P2 were tested for inhibition in this assay in thepresence of constant concentrations of 250 pM GDF8 or 250 pM GDF11, andno inhibition was observed after incubation of the cells with up to 150nM of the tested activin A antibodies; GDF8 and GDF11 alone in thisassay exhibited EC50 values of 124pM and 166pM, respectively. These datademonstrate that the Activin A antibodies H4H10446P2, H4H10430P,H4H10429P and H4H10436P2 do not functionally inhibit GDF8 or GDF11.

Example 7. Stimulation of Skeletal Muscle Hypertrophy Using Activin AAntibodies

Skeletal muscle hypertrophy induced by administration of amyostatin-specific antagonist, the anti-GDF8 antibody H4H1657N2 (see US2011-0293630 A1, hereby incorporated by reference in its entirety), or acombination of H4H1657N2 and different anti-Activin A antibodies, wasevaluated in CB17 SCID mice. The extent of hypertrophy was measuredrelative to treatment with an isotype-matched control antibody. Alsoincluded in these studies was treatment with the extracellular domain ofhuman ActRIIB, produced with a C-terminal human IgG1 Fc domain (Control4, SEQ ID No: 227). Control 4 has been previously shown to induce musclehypertrophy in vivo and also to bind and block the activity of multipleTGF-beta family member ligands (Souza, TA et al. Mol Endocrinol22:2689-702 (2008); Lee, SJ et al. Proc Natl Acad Sci U.S.A.102(50):18117-22 (2005)).

A total of eight anti-Activin A antibodies of the invention and Control1 were tested in combination with H4H1657N2 or alone in eight studies,in comparison to isotype control, Control 4, H4H1657N2 alone, or Control2 (an anti-Activin RIIB antibody having VH/VL of the antibody MOR08159described in US 2010/0272734 A1) treatment groups. For the studies, maleCB17 SCID mice (Taconic, #CB17SC-M) of approximately 10 weeks of agewere divided evenly according to body weight into 6 groups of 5 mice.Groups of mice were treated in each study as described in Table 13.

TABLE 13 Antibodies and controls tested in in vivo muscle hypertrophystudies Study # Samples Tested Dosage 1 Dosing interval of dosage 1Dosage 2 Dosing interval of dosage 2 1 Isotype Control 10 mg/kg days 0,3, and 7 8 mg/kg day 14 H4H1657N2 10 mg/kg days 0, 3, and 7 8 mg/kg day14 Control 4 10 mg/kg days 0, 3, and 7 8 mg/kg day 14H4H10423P+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, and 7 8 mg/kg+8 mg/kgday 14 H4H10432P2+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, and 7 8 mg/kg+8mg/kg day 14 H4H10442P2+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, and 7 8mg/kg+8 mg/kg day 14 2 Isotype Control 10 mg/kg days 0, 3, and 7 8 mg/kgday 14 H4H1657N2 10 mg/kg days 0, 3, and 7 8 mg/kg day 14 Control 4 10mg/kg days 0, 3, and 7 8 mg/kg day 14 H4H10429P+H4H1657N2 10 mg/kg+10mg/kg days 0, 3, and 7 8 mg/kg+8 mg/kg day 14 H4H10436P2+H4H1657N2 10mg/kg+10 mg/kg days 0, 3, and 7 8 mg/kg+8 mg/kg day 14H4H10440P2+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, and 7 8 mg/kg+8 mg/kgday 14 3 Isotype Control 10 mg/kg days 0, 3, 7, and 14 N/A H4H1657N2 10mg/kg days 0, 3, 7, and 14 Control 4 10 mg/kg days 0, 3, 7, and 14H4H10446P2+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, 7, and 14H4H10430P+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, 7, and 14 4 IsotypeControl 25 mg/kg days 0, 3, 7, and 14 N/A H4H1657N2 10 mg/kg days 0, 3,7, and 14 H4H10430P 10 mg/kg days 0, 3, 7, and 14 H4H10430P+H4H1657N2 2mg/kg+10 mg/kg days 0, 3, 7, and 14 H4H10430P+H4H1657N2 10 mg/kg+10mg/kg days 0, 3, 7, and 14 H4H10430P+H4H1657N2 25 mg/kg+10 mg/kg days 0,3, 7, and 14 5 Isotype Control 25 mg/kg days 0, 3, 7, and 14 N/AH4H1657N2 10 mg/kg days 0, 3, 7, and 14 H4H10446P2 10 mg/kg days 0, 3,7, and 14 H4H10446P2+H4H1657N2 2 mg/kg+10 mg/kg days 0, 3, 7, and 14H4H10446P2+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, 7, and 14H4H10446P2+H4H1657N2 25 mg/kg+10 mg/kg days 0, 3, 7, and 14 6 IsotypeControl 10 mg/kg days 0, 3, 7, 14, and 21 N/A H4H1657N2 10 mg/kg days 0,3, 7, 14, and 21 Control 4 10 mg/kg days 0, 3, 7, 14, and 21 Control 110 mg/kg days 0, 3, 7, 14, and 21 Control 1+H4H1657N2 10 mg/kg+10 mg/kgdays 0, 3, 7, 14, and 21 7 Isotype Control 10 mg/kg days 0, 3, 7, and 14N/A Control 4 10 mg/kg days 0, 3, 7, and 14 Control 2 25 mg/kg days 0,3, 7, and 14 H4H10430P+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, 7, and 14H4H10446P2+H4H1657N2 10 mg/kg+10 mg/kg days 0, 3, 7, and 14 8 IsotypeControl 25 mg/kg days 0, 3, 7, and 14 N/A H4H1657N2 10 mg/kg days 0, 3,7, and 14 Control 4 25 mg/kg days 0, 3, 7, and 14 Control 2 25 mg/kgdays 0, 3, 7, and 14 H4H10423P+H4H1657N2 25 mg/kg+10 mg/kg days 0, 3, 7,and 14 H4H10430P+H4H1657N2 25 mg/kg+10 mg/kg days 0, 3, 7, and 14

For studies 1-5, 7, and 8, antibodies and Control 4 were administeredsubcutaneously at a dose of 10 mg/kg of each protein twice during thefirst week of the experiment (days 0 and 3) and once at a dose of 10mg/kg of each protein during the second week (day 7). A final dose ofantibody or Control 4 during the third week (day 14) was administeredsubcutaneously at 8 mg/kg for studies #1 and #2 or at 10 mg/kg forstudies #3 - #8 (Table 13). On day 21, mice were euthanized and totalbody weight for each mouse was measured. For study 6, antibodies wereadministered for previous studies 1-5 but the treatment was extended today 28 with an additional injection at day 21. The tibialis anterior(TA) and gastrocnemius (GA) muscles from each mouse were dissected andweighed. Tissue weights were normalized to the starting body weight, andthe mean percent change in weight over the mean weight of the isotypecontrol antibody treatment group was calculated. Results summarized inTables 14-21 are expressed as mean percent increase over isotype control± standard error of the mean.

TABLE 14 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 1 Isotype Control H4H1657N2 Control 4H4H10423P + H4H1657N2 H4H10432P2 + H4H1657N2 H4H10442P2 + H4H1657N2 Dose10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg + 10 mg/kg 10 mg/kg + 10 mg/kg 10mg/kg + 10 mg/kg Body Weight 0.00 + 0.91 10.99 ± 0.48 18.45 ± 0.89 13.36± 1.10 12.84 ± 0.98 12.09 ± 0.78 TA Muscle 0.00 ± 1.15 19.54 ± 2.6745.80 ± 1.47 32.03 ± 2.12 24.83 ± 2.95 40.76 ± 2.59 GA Muscle 0.00 ±0.89 26.46 ± 3.63 31.91 ± 1.40 27.58 ± 1.61 26.39 ± 1.87 30.62 ± 2.32

As shown in Table 14, in the first study, Control 4 induced significanthypertrophy in all muscles examined, with increases of 45.80 ± 1.47% inTA, and 31.91 ± 1.4% in GA weights as compared to the isotype controltreated mice. Treatment with H4H1657N2 alone also induced hypertrophy inTA (19.54 ± 2.67% increase) and GA (26.46 ± 3.63% increase) muscleweights, but it was less efficacious than Control 4. The combination ofH4H1657N2 + H4H10442P2 induced similar increases in average TA (40.76 ±2.59%) and GA (30.62 ± 2.32%) muscle weights as compared to mice treatedwith Control 4. The combination treatments H4H1657N2/H4H10423P andH4H1657N2/H4H10432P2 did not induce increases in average TA weights asgreat as those induced by the H4H16757N2/H4H10442P or the Control 4treatments.

TABLE 15 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 2 Isotype Control H4H1657N2 Control 4H4H10429P + H4H1657N2 H4H10436P2+ H4H1657N2 H4H10440P2 + H4H1657N2 Dose10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg + 10 mg/kg 10 mg/kg + 10 mg/kg 10mg/kg +10 mg/kg Body Weight 0.00 + 2.53 4.12 ± 2.19 11.22 ± 1.71 7.17 ±1.57 7.89 ± 0.37 1.89 ± 1.39 TA Muscle 0.00 ± 3.59 16.70 ± 2.73 43.47 ±2.37 34.14 ± 2.55 29.31 ± 1.59 14.55 ± 2.22 GA Muscle 0.00 ± 3.54 18.54± 3.48 29.24 ± 2.22 26.24 ± 3.11 26.55 ± 2.41 15.65 ± 2.66

As shown in Table 15, in the second study, Control 4 induced hypertrophyin all muscles examined, with increases of 43.47 ± 2.37% in average TAweight and 29.24 ± 2.22% in GA average muscle weight as compared withthe isotype control treated mice. In this study, treatment withH4H1657N2 alone also induced increases in TA and GA average muscleweights (16.7 ±2.73% and 18.54 ± 3.48%, respectively) as compared withthe isotype control treated mice, but these average increases were lessthan those observed for the Control 4 treatment group. The combinationtreatments H4H1657N2/H4H10429P and H4H1657N2/H4H10436P2 inducedincreases in average TA (34.14 ± 2.55% and 29.31 ± 1.59%, respectively)and average GA (26.24 ± 3.11% and 26.55 ± 2.41%, respectively),increases that were between the increases observed for either H4H1657N2or Control 4 alone. The combination H4H1657N2/H4H10440P2 did not induceincreases in TA or GA average weights as great as those induced by theother two combinations in this study or by the Control 4 treatment.

TABLE 16 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 3 Isotype Control H4H1657N2 Control 4H4H10446P2 + H4H1657N2 H4H10430P + H4H1657N2 Dose 10 mg/kg 10 mg/kg 10mg/kg 10 mg/kg + 10 mg/kg 10 mg/kg + 10 mg/kg Body Weight 0.00 + 2.001.43 ± 1.14 18.92 ± 3.53 10.90 ± 2.51 8.88 ± 1.58 TA Muscle 0.00 ± 2.1314.19 ± 3.19 39.90 ± 3.58 40.01 ± 3.67 28.30 ± 3.27 GA Muscle 0.00 ±1.62 15.73 ± 0.58 34.01 ± 2.87 31.29 ± 2.60 21.55 ± 2.30

As shown in Table 16, in the third study, Control 4 induced hypertrophyin all muscles examined, with increases of 39.90 ± 3.58% in average TAmuscle weight, and 34.01 ± 2.87% in average GA muscle weight as comparedwith the isotype control-treated mice. Treatment with H4H1657N2 alonealso induced increases in TA (14.19 ± 3.19%) and GA average muscleweight (15.73 ± 0.58%) as compared with the isotype control treatedmice, but these average increases were less than those observed for theControl 4 treatment group. The combination treatmentH4H1657N2/H4H10446P2 induced similar increases in TA (40.01 ± 3.67%) andGA (31.29 ± 2.60%) average muscle weights as for mice treated withControl 4. The combination treatment with H4H1657N2/H4H10430P inducedincreases in TA (28.30 ±_3.27%) and GA (21.55 ± 2.30%) average muscleweights that were between those observed for H4H1657N2 alone and theH4H1657N2/H4H10446P2 combination treatment.

TABLE 17 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 4 Isotype Control H4H1657N2 H4H10430PH4H10430P + H4H1657N2 H4H10430P + H4H1657N2 H4H10430P + H4H1657N2 Dose25 mg/kg 10 mg/kg 10 mg/kg 2 mg/kg +10 mg/kg 10 mg/kg + 10 mg/kg 25mg/kg + 10 mg/kg Body Weight 0.00 ± 0.57 9.89 ± 0.98 4.20 ± 1.00 14.53 +0.80 12.61 ± 1.81 13.78 ± 1.58 TA Muscle 0.00 ± 3.04 21.05 ± 2.64 7.83 ±2.74 39.02 ± 3.55 40.20 ± 2.48 44.92 ± 5.70 GA Muscle 0.00 ± 2.71 22.85± 2.28 8.86 ± 1.24 27.57 ± 1.26 22.46 ± 5.03 30.22 ± 2.97

As shown in Table 17, in the fourth study, H4H1657N2 induced hypertrophyin the muscles examined, with increase of 21.05 ± 2.64% in average TAmuscle weight and 22.85 ± 2.28% in average GA muscle weight as comparedwith the isotype control treated mice. In this study, treatment withH4H10430P alone slightly increased muscle weights as compared to theisotype control treated mice but the values were not statisticallysignificant. The combination treatment of H4H1657N2 and H4H10430P at 10mg/kg and 2 mg/kg, respectively, induced increases in TA (39.02 ± 3.55%)and GA (27.57 ± 1.26%) average muscle weights that were greater in TAmuscle than those observed for H4H1657N2 or H4H10430P alone. Thecombination treatment of H4H1657N2 and H4H10430P at 10 mg/kg and 10mg/kg, respectively, induced increases in TA (40.20 ± 2.48%) and GA(22.46 ± 5.03%) average muscle weights that were greater in TA musclethan those observed for H4H1657N2 or H4H10430P alone. The combinationtreatment of H4H1657N2 and H4H10430P at 10 mg/kg and 25 mg/kg,respectively, induced increases in TA (44.92 ± 5.70%) and GA (30.22 ±2.97%) average muscle weights that were greater in TA muscle than thoseobserved for H4H1657N2 or H4H10430P alone.

TABLE 18 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 5 Isotype Control H4H1657N2 H4H10446P2H4H10446P2 + H4H1657N2 H4H10446P2 + H4H1657N2 H4H10446P2 + H4H1657N2Dose 25 mg/kg 10 mg/kg 10 mg/kg 2 mg/kg +10 mg/kg 10 mg/kg + 10 mg/kg 25mg/kg + 10 mg/kg Body Weight 0.00 ± 1.23 10.94 ± 1.03 0.29 ± 1.33 14.26± 1.45 12.61 ± 1.26 16.31 ± 2.04 TA Muscle 0.00 ± 2.20 25.40 ± 1.35 3.70± 1.67 51.29 ± 4.20 49.64 ± 4.08 49.79 ± 5.46 GA Muscle 0.00 ± 2.9222.82 ± 1.97 2.70 ± 1.06 39.24 ± 3.08 35.56 ± 3.39 35.14 ± 3.49

As shown in Table 18, in the fifth study, H4H1657N2 induced hypertrophyin the muscles examined, with increase of 25.4 ±1.35% in average TAmuscle weight and 22.82 ±1.97% in average GA muscle weight as comparedwith the isotype control treated mice. In this study, treatment withH4H10446P2 alone induced a low level of muscle hypertrophy with increaseof 3.70 ±1.67% in average TA muscle weight and 2.70 ±1.06% in average GAmuscle weight as compared with the isotype control treated mice. Thecombination treatment of H4H1657N2 and H4H10446P2 at 10 mg/kg and 2mg/kg, respectively, induced increases in TA (51.29 ± 4.20%) and GA(39.24 ± 3.08%) average muscle weights that were greater than thoseobserved for H4H1657N2 or H4H10446P2 alone. The combination treatment ofH4H1657N2 and H4H10446P2, each at a 10 mg/kg dose, induced increases inTA (49.64 ± 4.08%) and GA (35.56 ± 3.39%) average muscle weights thatwere greater than those observed for H4H1657N2 or H4H10446P2 alone. Thecombination treatment of H4H1657N2 and H4H10446P2 at 10 mg/kg and 25mg/kg, respectively, induced increases in TA (49.79 ± 5.46%) and GA(35.14 ± 3.49%) average muscle weights that were greater than thoseobserved for H4H1657N2 or H4H10446P2 alone.

TABLE 19 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 6 Isotype Control Control 4 H4H1657N2 Control 1Control 1 1 + H4H1657N2 Dose 10 mg/kg 10 mg/kg 10 mg/kg 10 mg/kg 10mg/kg + 10 mg/kg Body Weight 0.00 ± 0.51 17.04 ± 2.90 8.92 ± 1.26 3.52 ±0.86 15.84 ± 0.75 TA Muscle 0.00 ± 2.15 47.34 ± 2.63 17.21 ± 2.97 4.54 ±2.25 30.06 ± 5.51 GA Muscle 0.00 ± 1.71 32.17 ± 3.81 21.57 ± 1.90 2.72 ±1.30 30.72 ± 3.64

As shown in Table 19, in the sixth study, Control 4 induced hypertrophyin all muscles examined, with increases of 47.34 ± 2.63% in average TAweight and 32.17 ± 3.81% in GA average muscle weight as compared withthe isotype control treated mice. In this study, treatment withH4H1657N2 alone also induced increases in TA and GA average muscleweights 17.21 ± 2.97% and 21.57 ± 1.90%, respectively, as compared withthe isotype control treated mice, but these average increases were lessthan those observed for the Control 4 treatment group. In this study,treatment with Control 1 alone induced a low level of muscle hypertrophywith increase of 4.54 ±2.25% in average TA muscle weight and 2.72+1.30%in average GA muscle weight as compared with the isotype control treatedmice. The combination treatment of H4H1657N2 and Control 1 at 10 mg/kgand 10 mg/kg, respectively, induced increases in TA (30.06 ± 5.51%) andGA (30.72 ± 3.64%) average muscle weights that were greater than thoseobserved for H4H1657N2 or Control 1 alone.

TABLE 20 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 7 Isotype Control Control 4 Control2H4H10430P + H4H1657N2 H4H10446P2 + H4H1657N2 Dose 10 mg/kg 10 mg/kg 25mg/kg 10 mg/kg + 10 mg/kg 10 mg/kg + 10 mg/kg Body Weight 0.00 ± 0.908.17 ± 3.30 19.18 ± 1.75 10.55 ± 1.48 11.67 ± 0.98 TA Muscle 0.00 ± 2.3034.43 ± 5.92 36.75 ± 3.88 33.13 ± 2.02 41.28 ± 2.76 GA Muscle 0.00 ±2.01 14.86 ± 3.65 26.41 ± 3.16 22.82 ± 1.34 29.21 ± 2.62

As shown in Table 20, in the seventh study, Control 4-inducedhypertrophy in all muscles examined, with increases of 34.43 ± 5.92% inaverage TA weight and 14.86 ± 3.65% in GA average muscle weight ascompared with the isotype control treated mice. In this study, treatmentwith Control 2 alone induced hypertrophy in the muscles examined, withincreases of 36.75 ± 3.88% in average TA weight and 26.41 ± 3.16% in GAaverage muscle weight as compared with the isotype control treated mice.The combination treatment H4H1657N2 and H4H10430P at 10 mg/kg and 10mg/kg, respectively, induced increases in TA (33.13 ± 2.02%) and GA(22.82 ± 1.34%) average muscle weights that were between increasesobserved for ActRIIB-Fc alone and Control 2 alone. The combinationtreatment H4H1657N2 and H4H10446P2 at 10 mg/kg and 10 mg/kg,respectively, induced increases in TA (41.28 ± 2.76%) and GA (29.21 ±2.62%) average muscle weights.

TABLE 21 Percent change in body and muscle weights compared to isotypecontrol treatment, Study 8 Isotype Control Control 4 H4H1657N2 Control 2H4H10423P + H4H1657N2 H4H10430P + H4H1657N2 Dose 25 mg/kg 25 mg/kg 10mg/kg 25 mg/kg 25 mg/kg +10 mg/kg 25 mg/kg + 10 mg/kg Body Weight 0.00 ±0.64 19.81 ± 0.90 8.64 ± 1.30 21.56 ± 1.29 10.45 ± 1.40 15.45 ± 1.18 TAMuscle 0.00 ± 2.72 53.74 ± 5.31 18.44 ± 2.30 39.90 ± 1.69 36.33 ± 3.6743.83 ± 1.56 GA Muscle 0.00 ± 0.76 39.39 ± 4.56 21.17 ± 1.72 25.87 ±2.72 28.18 ± 3.11 31.24 ± 1.90

As shown in Table 21, in the eighth study, Control 4 induced hypertrophyin all muscles examined, with increases of 53.74 ± 5.31% in average TAweight and 39.39 ± 4.56% in GA average muscle weight as compared withthe isotype control treated mice. In this study, treatment withH4H1657N2 alone also induced increases in TA and GA average muscleweights of 18.44 ± 2.30% and 21.17 ± 1.72%, respectively, as comparedwith the isotype control treated mice, but these average increases wereless than those observed for the Control 4 treatment group. In thisstudy, treatment with Control 2 alone induced hypertrophy in the musclesexamined, with increases of 39.90 ± 1.69% in average TA weight and 25.87± 2.72% in GA average muscle weight as compared with the isotype controltreated mice. The combination treatment H4H1657N2 and H4H10423P at 10mg/kg and 25 mg/kg, respectively, induced increases in TA (36.33 ±3.67%)and GA (28.18 ± 3.11%) average muscle weights as compared with theisotype control treated mice. The combination treatment H4H1657N2 andH4H10430P at 10 mg/kg and 25 mg/kg, respectively, induced increases inTA (43.83 ± 1.56%) and GA (31.24 ± 1.90%) average muscle weights thatwere between increases observed for Control 4 alone and Control 2 alone.

These studies show that administration of anti-Activin A antibodies witha myostatin inhibitor can further increase skeletal muscle hypertrophyto a significantly greater degree than treatment with a myostatininhibitor alone at the doses and injection frequencies tested..

Example 8. Blocking of Activin A Binding Using Activin A Antibodies

The ability of selected anti-Activin A antibodies to block theinteraction of Activin A with its receptors, ActRllB and ActRIIA, aswell as its endogenous antagonist, Follistatin, was determined using aBIACORE™ 3000 instrument. For this experiment, Control 4 (human ActRllBexpressed with a C-terminal human Fc tag (SEQ ID:227)), human ActRIIAexpressed with a C-terminal human Fc tag (hActRIIA-Fc; R&D Systems, #340-R2-100), or Follistatin-288 (R&D Systems, #5836-FS-025) wereamine-coupled to a BIACORE™ CM5 sensor surface. Activin A (R&D Systems,#338-AC) at a fixed concentration of 5 nM either alone or mixed withActivin A antibodies, hActRIIA-Fc, hActRIIB-Fc, or isotype controlantibody at a final concentration of 60 nM (12-fold molar excess overActivin A) was incubated at room temperature for 1 hour. Theantibody-Activin A mixtures were then injected over the amine-coupledControl 4, hActRIIA-Fc, or Follistatin-288 surfaces at a flow rate of 20uL/min. The binding signal (RU) was measured at 150 seconds after thestart of the injection, and this signal was subtracted by the measuredRU value for a negative control reference surface to determine thespecific binding signal. The percentage of free Activin A binding overthe receptor or antagonist surfaces in the presence of each anti-ActivinA antibody was calculated as the ratio of the observed specific bindingsignal divided by the specific binding signal from 5 nM Activin A in thepresence of no antibody.

TABLE 22 Blocking of Activin A Binding to Follistatin by anti-Activin AAntibodies Follistatin-288 surface (3000RU captured)-Normalized toActivin A (% bound RU w/ no inhibitor) mAb/protein concentration (nM)H4H10442P2 H4H10446P2 H4H10430P H4H10440P2 H4H10429P H4H10436P2H4H10423P Control 1 Control 3 hActRIIA-hFc Control 4 (hActRIIB-hFc)isotype (-) control 0 100 100 100 100 100 100 100 100 100 100 100 1000.94 73 77 79 76 97 78 120 83 172 156 169 100 1.88 46 54 59 57 80 61 12268 170 148 163 102 3.75 6 7 15 17 20 16 103 27 145 138 151 97 7.5 3 3 14 1 1 97 0 33 116 120 102 15 3 3 1 2 1 1 96 1 5 60 43 102 30 3 3 1 1 2 294 1 7 11 1 104 60 3 3 1 0 3 2 93 2 9 13 1 103

As shown in Table 22, 6 of the 7 anti-Activin A antibodies of theinvention tested and both Control 1 and Control 3 blocked the binding ofActin A to Follistatin-288 . One antibody of the invention, H4H10423P,did not prevent binding of Activin A to Follistatin-288. Control 4 andhActRIIA-Fc blocked the binding of Activin A to Follistatin-288 athigher concentrations.

TABLE 23 Blocking of Activin A Binding to hActRIIA-Fc by anti-Activin AAntibodies g hActRIIA-hFc surface (8000RU captured)-Normalized toActivin A (% bound RU w/ no inhibitor) mAb/protein concentration (nM)H4H10442P2 H4H10446P2 H4H10430P h4H10440P2 H4H10429P H4H10436P2H4H10423P Control 1 Control 3 hActRIIA-hFc Control 4 (hActRIIB-hFc)isotype (-) control 0.00 100 100 100 100 100 100 100 100 100 100 100 1000.94 114 111 81 75 87 75 112 82 207 236 276 109 1.88 114 115 62 52 66 55114 66 190 222 266 112 3.75 95 85 19 17 19 16 111 28 139 188 231 1107.50 105 94 3 6 1 2 110 1 32 128 160 115 15 113 108 2 4 1 2 112 1 1 5051 116 30 117 98 2 3 1 2 114 1 1 5 2 118 60 118 118 2 3 1 2 116 2 0 3 1119

As shown in Table 23, 4 of the 7 anti-Activin A antibodies of theinvention tested and both Control 1 and Control 3 blocked the binding ofhActRIIA-Fc to Activin A. Three antibodies of the invention, H4H10442P2,H4H10446P2, and H4H10423P, did not prevent binding of Activin A tohActRIIA-Fc. Control 4 and hActRIIA-Fc blocked the binding of Activin Ato hActRIIA-Fc.

TABLE 24 Blocking of Activin A Binding to hActRIIB-Fc by anti-Activin AAntibodies hActRIIB-hFc (Control 4) surface (4000RU captured)-Normalizedto Activin A (% bound RU w/ no inhibitor) mAb/protein concentration (nM)H4H10442P2 H4H10446P2 H4H10430P H4H10440P2 H4H10429P H4H10436P2H4H10423P Control 1 Control 3 hActRIIA-hFc Control 4 (hActRIIB-hFc)isotype (-) control 0.00 100 100 100 100 100 100 100 100 100 100 100 1000.94 110 107 80 79 87 80 93 85 135 131 149 105 1.88 106 105 62 58 67 6078 69 133 129 148 105 3.75 88 76 20 19 19 19 47 31 120 127 144 104 7.50103 95 4 7 2 3 42 2 33 113 130 107 15 115 115 3 4 2 2 42 2 2 56 51 11030 122 89 3 4 2 3 41 2 1 5 3 111 60 124 129 3 4 3 4 41 3 2 5 2 115

As shown in Table 24, 4 of the 7 anti-Activin A antibodies of theinvention tested and both Control 1 and Control 3 blocked the binding ofActivin A to hActRIIB-Fc. Two antibodies of the invention, H4H10442P2and H4H10446P2, did not prevent binding of Activin A to hActRIIB-Fc. Oneantibody of the invention, H4H10423P, demonstrated the ability topartial block the binding of Activin A to hActRIIB-Fc at higherconcentrations of antibody tested. Both hActRIIB-Fc and hActRIIA-Fcblocked the binding of Activin A to hActRIIB-Fc.

Example 9. Effects of H4H1657N2 on Muscle Mass and Exercise Performance

The effects of the anti-GDF8 antibody H4H1657N2 on muscle mass andexercise performance was evaluated in aged male C57BL/6 mice (19 monthsold).

Mice were randomized into four groups (n=6-8/group), a sedentary orexercise group receiving subcutaneous doses of H4H1657N2 or an isotypecontrol antibody (10 mg/kg) twice weekly for 21 days (6 injections).Mice in the exercise group were placed on an exercise regimen involvingone training session a day, consisting of 20 minutes on an Exer 6 Mtreadmill (Columbus Instruments, Columbus, OH) at 10 m/min with a 5°incline, five days a week for three consecutive weeks. At the end ofthree weeks of treatment, endurance was measured in all four groupsusing a treadmill exhaustion test. The data were analyzed with two-wayANOVA followed by Tukey HSD test. Muscle weights were reported asnormalized weights (i.e., muscle weights were normalized to the bodyweights measured at the start of the experiment). Results for quadricepsmuscle are provided in Table 25 as average % change for each group (±standard error of the mean) compared to the isotype control antibodygroup.

TABLE 25 Quadracept Muscle Weight Change Isotype Control (Sedentary)H4H1657N2 (Sedentary) Isotype Control + Exercise H4H1657N2 + ExerciseQuad Weight 0.00 ± 2.72 15.77 ± 2.73 9.85 ± 3.57 17.66 ± 3.24 % Changefrom isotype control. Means ± SEM are shown.

As seen in Table 25, H4H1657N2 treatment resulted in significantincreases in the mass of quadriceps muscles (p<0.01 significance overisotype control for both H4H1657N2 groups). Increases in hindlimb musclegroup weights (TA, GA,) were seen in exercised (17.4%, 12.5%,respectively) and sedentary (14.1%, 11.6%, respectively) aged mice,compared with an isotype control antibody. A slight increase in muscleweight was observed between exercised and sedentary aged mice thatreceived isotype control antibody, but it was not statisticallysignificant(Table 25).

The effects of H4H1657N2 treatment on exercise endurance was alsoexamined in 19 month old male C57BL/6 mice (Table 26).

TABLE 26 Endurance Testing Isotype Control (Sedentary) H4H1657N2(Sedentary) Isotype Control + Exercise H4H1657N2 + Exercise Time Ranuntil Exhaustion (min) 27.94 ± 4.12 28.54 ± 6.10 50.26 ± 8.56 73.23 ±4.68 Distance Ran until Exhaustion (m) 428.42 ± 71.91 535.99 ± 155.61930.06 ± 179.78 1366.65 ± 95.91

In exercised aged mice, H4H1657N2 also induced significant increases inendurance, as measured by treadmill running time (73.2 min versus 50.2min) and distance (1.33 km versus 0.93 km), compared with the isotypecontrol group (Table 26). However, in sedentary mice, H4H1657N2 did notsignificantly increase endurance compared with the isotype controlgroup.

As in the muscle weight study, H4H1657N2 induced significant increasesin endurance, as measured by treadmill running time and distance, in theexercised mice only, but not in the sedentary mice. These results showthat H4H1657N2 increases physical performance outcomes when combinedwith exercise training.

Example 10. Effects of H4H1657N2 on Skeletal Muscle Mass and IsometricForce in Mice

The ability of H4H1657N2 to induce skeletal muscle hypertrophy wasassessed in vivo in 9 week old male C57BL/6 mice.

Repeated subcutaneous doses of H4H1657N2 or an isotype control antibody,at either 10 or 30 mg/kg, were administered twice weekly for 3 weeks(n=6). H4H1657N2 treatment for 21 days produced increases in body weightof 4.7 ± 2.3% (n.s.) and 7.1 ± 1.5% (n.s.), respectively, compared tomice receiving isotype control administered at equal doses. Individualmuscle weights were increased as follows compared to isotype control (10mg/kg & 30 mg/kg): Tibialis anterior (19.4 ± 4.9%** & 20.6± 1.5%**),Gastrocnemius: (14.9 ± 2.9%** & 25.3 ± 1.9%***), and Quadriceps (17.7 ±3.6%* & 26.2 ± 3.8%**). (All stats by One Way ANOVA with Tukey’s posthoc test [* p<0.05; ** p<0.01; *** p<0.001; n.s. = not statisticallydifferent].)

The increase in Tibialis anterior (TA) muscle mass was accompanied by anincrease in ex vivo isometric force, indicating the ability to maintainboth muscle function and mass. Mice previously treated with repeatedsubcutaneous doses of H4H1657N2 or isotype control antibody (at 10 mg/kgadministered twice weekly for 3 weeks, n= 6 per group) were individuallyanesthetized and maintained under Isoflurane gas while the TA muscle wasexcised placed in a oxygenated lactated ringers bath constantlymaintained at 25° C. The superior end of the TA was firmly tied to asubmerged stanchion in the bath while the distal tendon was tied to 305Clever arm (Cambridge Systems). Optimal length was determined by slightlystretching the TA and then testing the force produced by a 1 Hzstimulation at a minimal voltage. TA muscles were repeatedly stretchedand stimulated until there was a decline in force and then relaxed tothe previous position. Voltage was then incrementally increased in aseries of 1 HZ stimulations to achieve maximal force output. Onceoptimal length and voltage had been determined, TA muscles werestimulated for 400 milliseconds at increasing frequencies (40-100 Hz) todetermine maximum tetanic force. TA muscles were given 2 minute restperiods between each tetanic stimulation.

TA muscles from mice treated with an isotype control antibody at 10mg/kg and 30 mg/kg dose for 21 days generated an average peak tetanicforce of 892.6 ± 37 and 906.1 ± 37.8, respectively. TA muscles from micetreated with H4H1657N2 generated an average peak tetanic force of 1041.3± 31.7 and 1003.3 ± 35.7 mN, respectively. These force values representincrease of 16.7%* (10 mg/kg) and 10.7%n.s (30 mg/kg) in average peaktetanic force compared to isotype control (FIG. 2A). The overall drugeffect of H4H1657N2 treatment on peak tetanic force was statisticallydifferent from isotype control at both 10 mg/kg and 30 mg/kg doses (10mg/kg dose shown in FIG. 2B). (FIG. 2A: statistical analysis by One WayANOVA with Tukey’s post hoc test [* p<0.05; n.s. = not statisticallydifferent]. FIG. 2B: statistical analysis by Two way ANOVA and Sidakspost hoc test [p>0.0001].)

Example 11. H4H1657N2 Improves the Recovery From Hind Limb Suspension(HLS)-Induced Atrophy

The effect of H4H1657N2 on skeletal muscle mass during the recoveryphase from 7 days of hindlimb suspension (HLS) induced atrophy wasassessed in one-year old C57BL/6 male mice.

At day 0, eighteen mice were suspended by the tail so that both hindlegs could not touch the ground for the duration of 7 days. Mice werehoused in special cages with free access to food and water.Concurrently, one additional group of six mice was left in normal cagingand served as a control (Non-HLS control). At day 7, the suspended micewere taken down and randomized by percentage of body weight lost duringHLS into three groups (n=6 each). At day 7, the muscle weights from theNon-HLS control group and one HLS group (HLS group) were taken to assessthe percentage of atrophy in response to HLS. The two remaining HLSgroups (n=6 each) were allowed to recover for 8 days (i.e., day 7through day 15 of the experiment) in normal caging and treatedsubcutaneously with 10 mg/kg doses of either H4H1657N2 or an isotypecontrol on days 7 and 10 (i.e., after zero days and 3 days of recovery)(HLS+7Rec+H4H1657N2 and HLS+7rec+Isotype Control, respectively). At day15 (i.e., after 8 days of recovery), muscle weights were taken to assessthe percentage of recovery after HLS-induced atrophy.

As seen in FIG. 3B, seven days of HLS resulted in significant loss ofmass in both tibialis anterior (TA) and gastrocnemius (GA) (HLS group),as compared to the Non-HLS control group (-13.7%* and -14.8%*respectively). After 8 days of recovery, the HLS+7rec+Isotype Controlgroup maintained losses in TA and GA muscle mass (-6.3% and -7.5%) ascompared to the Non-HLS control group, whereas the HLS+7Rec+H4H1657N2group showed gains in mass (4.7% and 5%) as compared to the Non-HLSgroup.

When comparing the two recovery groups (i.e., HLS+7Rec+H4H1657N2 versusHLS+7rec+Isotype Control), the effects of H4H1657N2 on TA and GA masswere not statistically different from the effects seen with the isotypecontrol antibody. However, while the HLS+7rec+Isotype Control group’smuscle mass was not statistically different from the HLS group or theNon-HLS control group, the HLS+7Rec+H4H1657N2 group had statisticallylarger TA and GA mass when compared to the HLS group. (All stats by OneWay ANOVA with Tukey’s post hoc test [* p<0.05 vs. No HLS; ## p<0.01 vs.HLS.)

Example 12. Inhibition of BMP Receptor Type I and II Activation ByAnti-Activin A Antibodies and ActRIIB-Fc

Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily andare involved in regulation of many physiological processes by activatingreceptor complexes on the cell surface that are composed of BMP receptortypes I and II. Activation of receptors leads to phosphorylation of SMADproteins and transcriptional activation of ligand-responsive genes.

A bioassay was developed to detect the regulation of BMP signaling inW-20-17 cells, a mouse bone marrow stromal cell line previously shown tobe responsive to BMP2. The cells were engineered to stably express aluciferase reporter (i.e., BMP-responsive element(BRE(2X)-luciferase-IRES-GFP)), and sorted for high expression of GFP.The resultant stable cell line is referred to as W-20-17/BRE-luc and wasmaintained in 10% FBS, DMEM, Pen/Strep, and 200 µg/ml G418. These cellswere used to measure BMP activation and the inhibition of thisactivation by anti-Activin A antibodies and ActRIIB-hFc (Control 4, SEQID No:227).

The ability of four anti-Activin A antibodies and ActRIIB-hFc to inhibitBMP signaling was evaluated using the W-20-17/BRE-luc cell line. For thebioassay, W-20-17/BRE-luc cells are seeded onto 96-well assay plates at10,000 cells/well and incubated at 37° C. and 5% CO2 overnight. The nextday, BMP2, BMP4, BMP6, BMP9 or BMP10 were serially diluted at 1:3 andadded to cells from 100 nM to 0.002 nM (including no BMP control fordose responses). For inhibition of BMPs by anti-Activin A antibodies orActRIIB-hFc, antibodies or ActRIIB-hFc were serially diluted at 1:3 from1000 nM to 0.02 nM (including no antibody, control antibody, or negativecontrol for ActRIIB-hFc (i.e., an irrelevant protein tagged with hFc,“Control Protein”)) and added to cells along with 100pM BMP2, 100pMBMP4, 10 nM BMP6, 800pM BMP9 or 4 nM BMP10, as indicated. Luciferaseactivity was detected after 5.5 hrs of incubation in 37° C. and 5% CO2with Victor X (Perkin Elmer) and the results were analyzed using usingnonlinear regression (4-parameter logistics) with Prism 5 software(GraphPad).

As shown in Table 27 below, H4H10446P2 and H4H10430P did not inhibit ofany of the BMPs tested, whereas the other Activin A antibodies tested(H4H10429 and H4H10436P2) and ActRIIB-hFc all showed some inhibition ofsome of the BMPs. H4H10429P showed inhibition of BMP9 and BMP10 withIC₅₀ values of 8.1 nM and 3.5 nM, respectively, but did not inhibitBMP2, BMP4 and BMP6. H4H10436P2 showed weak inhibition of BMP2 and BMP4at highest concentrations of the antibody and inhibition of BMP10 withan IC₅₀ value of >100 nM, but did not show any inhibition of BMP6 andBMP9. ActRIIB-hFc showed inhibition of BMP9 and BMP10 with IC₅₀ valuesof 2 nM and 1 nM but did not inhibit BMP2, BMP4, and BMP6. Neither ofthe control molecules (i.e., an isotype control antibody (Control mAb)and irrelevant protein tagged with hFc (Control Protein)), were seen toinhibit any of the BMPs, whereas BMP2, BMP4, BMP6, BMP9, or BMP10 alone(i.e., without antibodies or hFc-tagged proteins) activated theW-20-17/BRE-luc cells with EC₅₀ values of 34pM, 63pM, 4.5 nM, 260pM, and2.5 nM, respectively.

TABLE 27 Inhibition by anti-Activin A antibodies and ActRIIb-hFc of BMPsin W-20-17/BRE-luc cells LIgands BMP2 BMP4 BMP6 BMP9 BMP10 EC50 [M]3.4E-11 6.3E-11 4.5E-09 2.6E-10 2.5E-09 Constant BMP 100 pM 100 pM 10 nM800 pM 4 nM Antibodies IC50 [M] IC50 [M] IC50 [M] IC50 [M] IC50 [M]H4H10446P2 No Inhibition No Inhibition No Inhibition No Inhibition NoInhibition H4H10430P No Inhibition No Inhibition No Inhibition NoInhibition No Inhibition H4H10429P No Inhibition No Inhibition NoInhibition 8.1E-09 3.5E-09 H4H10436P2 Weak (31% inhibition at 1uM) Weak(51% inhibition at 1uM) No Inhibition No Inhibition > 1.0E-07ActRIIB-hFc No Inhibition No Inhibition No Inhibition 2.0E-09 1.0E-09Control mAb No Inhibition No Inhibition No Inhibition No Inhibition NoInhibition Control Protein No Inhibition No Inhibition No Inhibition NoInhibition No Inhibition

Example 13. Treatment With Anti-Activin A Antibody H4H10446P2 ReducesRenal Fibrosis In Vivo

The effect of a specific anti-activin A antibody of the invention,H4H10446P2, on renal fibrosis was determined in an unilateral ureteralobstruction (UUO) mouse model of renal fibrosis. The UUO model wasdeveloped by complete ligation of the left ureter while keeping theright kidney function intact. Briefly, UUO was performed in mice underKetamine/Xylazine anesthesia, whereby the left ureter was accessed viaflank incision, and two ligatures were placed on the proximal one-thirdof the ureter using 5-0 silk thread at 5 mm apart. Sham surgeries weredone in a similar fashion without placing any ligatures on the ureter.In this model, severe fibrosis develops in the kidney within 14 daysfollowing UUO, which has been assessed by measuring kidney collagen bydirectly measuring the amount of hydroxyproline in the sample, which isreferred to as the hydroxyproline method. Hydroxyproline is a specificcomponent of collagens, and represents approximately 14.4% of the aminoacid composition of collagen in most mammalian tissues (Cochrane et al.,J Am Soc Nephrol 16:3623-30 (2005)). To measure collagen content via thehydroxyproline method, first frozen kidney samples were dried overnightusing a vacuum chamber. Dried kidney tissue samples were thenhomogenized in an ice-cold NaCl/NaHCO₃ solution and were then hydrolyzedusing 6 M HCl. The samples were subsequently dried using a vacuumcentrifuge, and then were rehydrated using 0.1 M HCl. The hydroxyprolinein the rehydrated samples was oxidized with 300 mM Chloramine T (Sigma,# 857319) and Ehrlich’s reagent [3.5 M p-dimethylaminobenzaldehyde (FW:149.19, Sigma, # 39070) in 60% perchloric acid (Sigma, # 311413)] wasthen added to develop the color. Finally, using a spectrophotometer,absorbance of the samples was measured at 558 nm and this was comparedto hydroxyproline standards (Sigma, # H5534) of known concentration, todetermine the kidney hydroxyproline content. The measured hydroxyprolinevalue was then multiplied by a factor of 6.94 to determine the collagenvalue. Fourteen days following UUO, dry kidney weight decreases as aresult of parenchymal damage. Sham (n = 10) or UUO (n = 20) surgerieswere performed on 16-week old male C57BL/6 mice (Taconic farms, Inc.).Mice, which underwent UUO surgery, were then divided into two groups.Each UUO group received a subcutaneous injection of either H4H10446P2(40 mg/kg, n = 10) or an isotype control antibody (40 mg/kg, n = 10),which does not bind to any known mouse protein, starting a day beforethe surgeries, and on 1, 3, 6, 8, 10, and 13 days after the surgery. Themice that underwent the sham surgery received vehicle (sterile PBS)during this time using the same schedule as the UUO groups. All the micewere sacrificed on day 14 following surgery. The kidney weights weremeasured, and the kidneys were flash-frozen using liquid nitrogen, andkept at -80° C. until the collagen content was measured. Kidney collagencontent was measured using the hydroxyproline method, and then expressedas either total kidney collagen (µg) or kidney collagen normalized tokidney weight (µg/mg of dry weight). Statistical analysis was done usingOne-Way ANOVA with Turkey’s multiple comparison test. The resultsincluding summarizes total kidney collagen, normalized kidney collagen,and dry kidney weights for each treatment group were expressed as mean ±SEM in Table 28 below.

TABLE 28 Total Kidney collagen, Normalized Kidney Collagen, and DryKidney Weight in each group (mean ± SEM) Treatment Group Total KidneyCollagen (µg) Normalized Kidney Collagen (µg/mg of tissue dry weight)Dry Kidney Weight (g) Sham + Vehicle 429.6 ± 25.93 8.16 ± 0.29 0.0524 ±0.002 UUO + Isotype Control 980.7 ± 50.48 25.07 ± 0.86 0.0396 ± 0.0027UUO + H4H10446P2 730.7 ± 48.02 17.48 ± 0.79 0.0422 ± 0.0029

As shown in Table 28, both total kidney collagen and kidney collagennormalized to kidney weight was significantly increased in UUO micecompared to sham-operated mice. UUO mice treated with H4H10446P2exhibited significant reduction in both total kidney collagen and kidneycollagen normalized to kidney weight (approximately 45% reduction infibrotic collagen) compared to isotype control antibody treated UUOmice, indicating the anti-activin A antibody lead to decreased fibrosisin the kidney. UUO mice treated with H4H10446P2 exhibited an increase indry kidney weight compared to the isotype control antibody treated UUOmice, indicating preservation of parenchyma in the anti-activin Aantibody treated mice.

Example 14. Effects of H4H10446P2 on Body Weight and Muscle Mass in MiceOverexpressing Activin A

To assess the efficacy of H4H10446P2 in neutralizing elevated levels ofActivin A in mice, Activin A was over-expressed in C57BL/6 mice (10weeks-old) by hydrodynamic delivery (HDD) of a DNA construct encodingfull-length Activin A. Mice were randomized into three groups(n=5-6/group); one was injected with a mixture of saline/2.5 µg of a DNAconstruct control in presence of an isotype control antibody, and twogroups were injected with a mixture of saline/2.5 µg of a DNA constructcontaining Activin A in presence of an isotype control antibody orH4H10446P2. DNA constructs were injected on day 0, and antibodies wereadministered on days 0 and 4 at 2.5 mg/kg (2 injections) for 7 days.Muscle weights were reported as normalized weights (i.e., muscle weightswere normalized to the body weights measured at the start of theexperiment). Results for body weights are shown as average change fromstarting body weights. Results for tibialis anterior (TA) andgastrocnemius (GA) muscles are shown in FIG. 4 as average percent changefor each group (± standard error of the mean) compared to the HDDdelivery of a construct control + isotype control antibody group. Thedata were analyzed with one or two-way ANOVA followed by Tukey HSD test.

As seen in FIG. 4 , seven days after HDD, delivery of Activin A in micetreated with an isotype control antibody resulted in significantdecreases in body weights (-10.81 ± 2.46%) and the mass of tibialis andgastrocnemius muscles (of -13.96 ± 1.85% and of -10.34 ± 1.51%,respectively) (p<0.01 significance over isotype control). Delivery ofActivin A in mice treated with H4H10446P2 resulted in a significantattenuation of body weights (-1.49 ± 1.98%) and the mass of tibialis andgastrocnemius muscles at the end of seven days of treatment (of -2.57 ±1.26% and of -1.77 ± 2.42%, respectively).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof that specifically binds Activin A and comprises (a) thecomplementarity determining region domains (CDRs) of a heavy chainvariable region (HCVR) having the amino acid sequence of SEQ ID NO: 138;and (b) the CDRs of a light chain variable region (LCVR) having theamino acid sequence of SEQ ID NO:
 146. 2. The isolated antibody orantigen-binding fragment thereof of claim 1, wherein the isolatedantibody or antigen-binding fragment thereof specifically binds ActivinA with a K_(D) of less than about 5 pM as measured in a surface plasmonresonance assay at 25° C.
 3. The isolated antibody or antigen-bindingfragment thereof of claim 1, wherein the isolated antibody orantigen-binding fragment thereof specifically binds Activin A with abinding association equilibrium constant (K_(a)) of less than about 500nM.
 4. The isolated antibody or antigen-binding fragment thereof ofclaim 1, wherein the antibody or antigen-binding fragment thereof blocksbinding of at least one Activin A receptor to Activin A.
 5. The isolatedantibody or antigen-binding fragment thereof of claim 1, wherein theantibody or antigen-binding fragment thereof blocks activation of atleast one Activin A receptor by Activin A.
 6. The isolated antibody orantigen-binding fragment thereof of claim 5, wherein the antibody orantigen-binding fragment thereof does not significantly block binding ofActivin A to an Activin Type II receptor.
 7. The isolated antibody orantigen-binding fragment thereof of claim 4, wherein the antibody orantigen-binding fragment thereof blocks Activin A binding to an ActivinA receptor with an IC₅₀ value of less than about 80 pM as measured in anin vivo receptor/ligand binding bioassay at 25° C.
 8. The isolatedantibody or antigen-binding fragment thereof of claim 7, wherein theantibody or antigen-binding fragment thereof blocks Activin A binding toan Activin A receptor with an IC₅₀ value of less than about 60 pM asmeasured in an in vivo receptor/ligand binding bioassay at 25° C.
 9. Theisolated antibody or antigen-binding fragment thereof of claim 1,wherein the antibody or antigen-binding fragment thereof inhibitsbinding of Activin A to an Activin A receptor selected from the groupconsisting of Activin Type IIA receptor (ActRIIA), Activin Type IIBreceptor (ActRIIB), and Activin Type 1 receptor.
 10. The isolatedantibody or antigen-binding fragment thereof of claim 1, wherein theantibody or antigen-binding fragment thereof inhibits Activin A-mediatedactivation of SMAD complex signaling.
 11. The isolated antibody orantigen-binding fragment thereof of claim 1, wherein the antibody orantigen-binding fragment thereof competes for binding to Activin A witha reference antibody comprising a heavy chain variable region(HCVR)/light chain variable region (LCVR) sequence pair selected fromthe group consisting of SEQ ID NOs: 2/10, 162/146, and 194/146.
 12. Theisolated antibody or antigen-binding fragment thereof of claim 1,wherein the antibody or antigen-binding fragment thereof binds to thesame epitope on Activin A as a reference antibody comprising anHCVR/LCVR sequence pair selected from the group consisting of SEQ IDNOs: 2/10, 162/146, and 194/146.
 13. (canceled)
 14. The isolatedantibody or antigen-binding fragment thereof of claim 1, wherein theantibody or antigen-binding fragment comprises the HCVR/LCVR amino acidsequence pair of: SEQ ID NOs: 138/146.
 15. The isolated antibody orantigen-binding fragment thereof of claim 14, wherein the antibody orantigen-binding fragment thereof comprisesHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences_of: SEQ ID NOs: 140-142-144-148-150-152. 16.(canceled)
 17. (canceled)
 18. A pharmaceutical composition comprisingthe antibody or antigen-binding fragment of claim 1, and apharmaceutically acceptable carrier or diluent.
 19. A method forincreasing muscle mass or strength in a subject, the method comprisingadministering to the subject the pharmaceutical composition of claim 18.20. The pharmaceutical composition of claim 18, further comprising agrowth and differentiation factor-8 (GDF8) antagonist.
 21. Thepharmaceutical composition of claim 20, wherein GDF8 antagonist isselected from the group consisting of a GDF8-inhibiting fusion protein,an anti- GDF8 antibody, and an antigen-binding fragment of an anti-GDF8antibody.
 22. The method of claim 19, further comprising theadministration of a GDF8 antagonist. wherein the GDF8 antagonist is ananti-GDF8 antibody or antigen-binding fragment thereof.
 23. The methodof claim 22, wherein the GDF8 antagonist is an anti-GDF8 antibody orantigen-binding fragment thereof comprising the heavy chaincomplementarity determining regions (HCDRs) of a HCVR comprising SEQ IDNO:217, and the light chain complementarity determining regions (LCDRs)of a LCVR comprising SEQ ID NO:221.
 24. The method of claim 22, whereinthe GDF8 antagonist is an anti-GDF8 antibody or antigen-binding fragmentthereof comprising: a) three HCDRs comprising SEQ ID NO:218, SEQ IDNO:219, and SEQ ID NO:220, and b) three LCDRs comprising SEQ ID NO:222,SEQ ID NO:223, and SEQ ID NO:224.
 25. A method for increasing musclemass or strength in a subject, the method comprising administering tothe subject the pharmaceutical composition of claim
 20. 26. A method forincreasing muscle mass or strength in a subject, the method comprisingadministering to the subject an antigen-binding molecule comprising anActivin A-specific binding domain and a GDF8-specific binding domain,wherein the Activin A-specific binding domain comprises an HCVR and aLCVR, wherein the HCVR comprises: (a) the CDRs of a HCVR having theamino acid sequence of SEQ ID NO: 138; and the LCVR comprises (b) theCDRs of a LCVR having the amino acid of SEQ ID NO:
 146. 27. (canceled)28. The method of claim 26, wherein the GDF8-specific binding domaincomprises a HCVR and a LCVR.
 29. The method of claim 26, wherein theHCVR comprises: (a) a HCVR having the amino acid sequence of SEQ ID NO:138; and the LCVR comprises (b) a LCVR having the amino acid sequence ofSEQ ID NO:
 146. 30. The method of claim 28, wherein the HCVR of theGDF8-specific binding domain comprises three heavy chain complementaritydetermining regions (HCDRs) comprising SEQ ID NO:218, SEQ ID NO:219, andSEQ ID NO:220, and wherein the LCVR of the GDF8-specific binding domaincomprises three light chain complementarity determining regions (LCDRs)comprising SEQ ID NO:222, SEQ ID NO:223, and SEQ \ID NO:224. 31.(canceled)
 32. The method of claim 26, wherein the antigen-bindingmolecule is a bispecific antibody.
 33. A method for treating, preventingor ameliorating a disease or disorder characterized by decreased musclemass or strength, the method comprising administering to a subject inneed thereof an Activin A-specific binding protein, wherein the ActivinA-specific binding protein comprises (a) the complementarity determiningregion domains (CDRs) of a heavy chain variable region (HCVR) having theamino acid sequence of SEQ ID NO: 138; and (b) the CDRs of a light chainvariable region (LCVR) having the amino acid sequence of SEQ ID NO: 146.34. The method of claim 33 further comprising administering to thesubject in need thereof a GDF8-specific binding protein.
 35. The methodof claim 34, wherein the disease or disorder characterized by decreasedmuscle mass or strength is selected from the group consisting ofsarcopenia, cachexia, muscle injury, muscle wasting/atrophy, cancer,obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy,amyotrophic lateral sclerosis, Parkinson’s disease, osteoporosis,osteoarthritis, osteopenia, and a metabolic syndrome.
 36. The method ofclaim 35, wherein the cachexia is idiopathic or is cachexia secondary toanother condition.
 37. The method of claim 36, wherein the condition iscancer, chronic renal failure, or chronic obstructive pulmonary disease.38. The method of claim 35, wherein the muscle wasting/atrophy is causedby or associated with a condition selected from the group consisting ofdisuse, immobilization, bed rest, injury, medical treatment, surgicalintervention and by necessity of mechanical ventilation.
 39. The methodof claim 38, wherein the surgical intervention is selected from thegroup consisting of hip fracture, hip replacement, and knee replacement.40. The method of claim 35, wherein the metabolic syndrome includes adisease or disorder selected from the group consisting of diabetes,obesity, nutritional disorders, organ atrophy, chronic obstructivepulmonary disease, and anorexia.
 41. A method for treating, preventingor ameliorating a disease or disorder characterized by decreased musclemass or strength, the method comprising administering to a subject inneed thereof an antigen-binding molecule comprising an ActivinA-specific binding domain and a GDF8-specific binding domain, whereinthe Activin A-specific binding domain comprises an HCVR and a LCVR,wherein the HCVR comprises: (a) the CDRs of a HCVR having the amino acidsequence of SEQ ID NO: 138; and the LCVR comprises (b) the CDRs of a LCVR having the amino acid of SEQ ID NO:
 146. 42. A method for treating,preventing or ameliorating a disease or disorder that is caused by,promoted by, exacerbated by, or aggravated by Activin A activity, themethod comprising administering to a subject in need thereof the ActivinA antibody or antigen-binding fragment thereof of claim
 1. 43. Themethod of claim 42, wherein the disease or disorder is renal fibrosis.44. The method of claim 42, wherein the disease or disorder is cachexia.